Publications by authors named "Peter M Burgers"

103 Publications

Novel insights into the mechanism of cell cycle kinases Mec1(ATR) and Tel1(ATM).

Crit Rev Biochem Mol Biol 2021 Oct 20;56(5):441-454. Epub 2021 Jun 20.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO, USA.

DNA replication is a highly precise process which usually functions in a perfect rhythm with cell cycle progression. However, cells are constantly faced with various kinds of obstacles such as blocks in DNA replication, lack of availability of precursors and improper chromosome alignment. When these problems are not addressed, they may lead to chromosome instability and the accumulation of mutations, and even cell death. Therefore, the cell has developed response mechanisms to keep most of these situations under control. Of the many factors that participate in this DNA damage response, members of the family of phosphatidylinositol 3-kinase-related protein kinases (PIKKs) orchestrate the response landscape. Our understanding of two members of the PIKK family, human ATR (yeast Mec1) and ATM (yeast Tel1), and their associated partner proteins, has shown substantial progress through recent biochemical and structural studies. Emerging structural information of these unique kinases show common features that reveal the mechanism of kinase activity.
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http://dx.doi.org/10.1080/10409238.2021.1925218DOI Listing
October 2021

The fidelity of DNA replication, particularly on GC-rich templates, is reduced by defects of the Fe-S cluster in DNA polymerase δ.

Nucleic Acids Res 2021 06;49(10):5623-5636

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA.

Iron-sulfur clusters (4Fe-4S) exist in many enzymes concerned with DNA replication and repair. The contribution of these clusters to enzymatic activity is not fully understood. We identified the MET18 (MMS19) gene of Saccharomyces cerevisiae as a strong mutator on GC-rich genes. Met18p is required for the efficient insertion of iron-sulfur clusters into various proteins. met18 mutants have an elevated rate of deletions between short flanking repeats, consistent with increased DNA polymerase slippage. This phenotype is very similar to that observed in mutants of POL3 (encoding the catalytic subunit of Pol δ) that weaken binding of the iron-sulfur cluster. Comparable mutants of POL2 (Pol ϵ) do not elevate deletions. Further support for the conclusion that met18 strains result in impaired DNA synthesis by Pol δ are the observations that Pol δ isolated from met18 strains has less bound iron and is less processive in vitro than the wild-type holoenzyme.
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http://dx.doi.org/10.1093/nar/gkab371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191807PMC
June 2021

Mechanism of auto-inhibition and activation of Mec1 checkpoint kinase.

Nat Struct Mol Biol 2021 01 9;28(1):50-61. Epub 2020 Nov 9.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO, USA.

In response to DNA damage or replication fork stalling, the basal activity of Mec1 is stimulated in a cell-cycle-dependent manner, leading to cell-cycle arrest and the promotion of DNA repair. Mec1 dysfunction leads to cell death in yeast and causes chromosome instability and embryonic lethality in mammals. Thus, ATR is a major target for cancer therapies in homologous recombination-deficient cancers. Here we identify a single mutation in Mec1, conserved in ATR, that results in constitutive activity. Using cryo-electron microscopy, we determine the structures of this constitutively active form (Mec1(F2244L)-Ddc2) at 2.8 Å and the wild type at 3.8 Å, both in complex with Mg-AMP-PNP. These structures yield a near-complete atomic model for Mec1-Ddc2 and uncover the molecular basis for low basal activity and the conformational changes required for activation. Combined with biochemical and genetic data, we discover key regulatory regions and propose a Mec1 activation mechanism.
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http://dx.doi.org/10.1038/s41594-020-00522-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855233PMC
January 2021

Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis.

J Biol Chem 2020 11 10;295(47):15883-15891. Epub 2020 Sep 10.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri USA. Electronic address:

Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.
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http://dx.doi.org/10.1074/jbc.RA120.015699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681027PMC
November 2020

Bypass of DNA interstrand crosslinks by a Rev1-DNA polymerase ζ complex.

Nucleic Acids Res 2020 09;48(15):8461-8473

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO, USA.

DNA polymerase ζ (Pol ζ) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases η, ζ and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based interstrand crosslink (ICL) with an 8-atom linker between the crosslinked bases. The Rev1-Pol ζ complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol ζ alone or Pol η. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol η, Pol ζ, and Rev1-Pol ζ. Rev1-Pol ζ, and particularly Pol ζ alone showed a tendency to stall before the ICL, whereas Pol η stalled just after insertion across the ICL. The stalling of Pol η directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol η providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol ζ directly past the ICL was observed, suggesting that the proposed function of Pol ζ as an extender DNA polymerase is also required for ICL repair.
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http://dx.doi.org/10.1093/nar/gkaa580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470978PMC
September 2020

Modeling cancer genomic data in yeast reveals selection against ATM function during tumorigenesis.

PLoS Genet 2020 03 18;16(3):e1008422. Epub 2020 Mar 18.

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.
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http://dx.doi.org/10.1371/journal.pgen.1008422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105138PMC
March 2020

The telomere-binding protein Rif2 and ATP-bound Rad50 have opposing roles in the activation of yeast Tel1 kinase.

J Biol Chem 2019 12 22;294(49):18846-18852. Epub 2019 Oct 22.

Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, Missouri 63110. Electronic address:

Tel1 is the ortholog of human ATM kinase and initiates a cell cycle checkpoint in response to dsDNA breaks (DSBs). Tel1 kinase is activated synergistically by naked dsDNA and the Mre11-Rad50-Xrs2 complex (MRX). A multisubunit protein complex, which is related to human shelterin, protects telomeres from being recognized as DSBs, thereby preventing a Tel1 checkpoint response. However, at very short telomeres, Tel1 can be recruited and activated by the MRX complex, resulting in telomere elongation. Conversely, at long telomeres, Rap1-interacting-factor 2 (Rif2) is instrumental in suppressing Tel1 activity. Here, using an reconstituted Tel1 kinase activation assay, we show that Rif2 inhibits MRX-dependent Tel1 kinase activity. Rif2 discharges the ATP-bound form of Rad50, which is essential for all MRX-dependent activities. This conclusion is further strengthened by experiments with a Rad50 allosteric ATPase mutant that maps outside the conserved ATP binding pocket. We propose a model in which Rif2 attenuates Tel1 activity at telomeres by acting directly on Rad50 and discharging its activated ATP-bound state, thereby rendering the MRX complex incompetent for Tel1 activation. These findings expand our understanding of the mechanism by which Rif2 controls telomere length.
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http://dx.doi.org/10.1074/jbc.RA119.011077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901328PMC
December 2019

The roles of fission yeast exonuclease 5 in nuclear and mitochondrial genome stability.

DNA Repair (Amst) 2019 11 21;83:102720. Epub 2019 Sep 21.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

The Exo5 family consists of bi-directional, single-stranded DNA-specific exonucleases that contain an iron-sulfur cluster as a structural motif and have multiple roles in DNA metabolism. S. cerevisiae Exo5 is essential for mitochondrial genome maintenance, while the human ortholog is important for nuclear genome stability and DNA repair. Here, we identify the Exo5 ortholog in Schizosaccharomyes pombe (spExo5). The activity of spExo5 is highly similar to that of the human enzyme. When the single-stranded DNA is coated with single-stranded DNA binding protein RPA, spExo5 become a 5'-specific exonuclease. Exo5Δ mutants are sensitive to various DNA damaging agents, particularly interstrand crosslinking agents. An epistasis analysis places exo5 in the Fanconi pathway for interstrand crosslink repair. Exo5 is in a redundant pathway with rad2, which encodes the flap endonuclease FEN1, for mitochondrial genome maintenance. Deletion of both genes lead to severe depletion of the mitochondrial genome, and defects in respiration, indicating that either spExo5 or spFEN1 is necessary for mitochondrial DNA metabolism.
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http://dx.doi.org/10.1016/j.dnarep.2019.102720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6990415PMC
November 2019

Complementary roles of Pif1 helicase and single stranded DNA binding proteins in stimulating DNA replication through G-quadruplexes.

Nucleic Acids Res 2019 09;47(16):8595-8605

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.

G-quadruplexes (G4s) are stable secondary structures that can lead to the stalling of replication forks and cause genomic instability. Pif1 is a 5' to 3' helicase, localized to both the mitochondria and nucleus that can unwind G4s in vitro and prevent fork stalling at G4 forming sequences in vivo. Using in vitro primer extension assays, we show that both G4s and stable hairpins form barriers to nuclear and mitochondrial DNA polymerases δ and γ, respectively. However, while single-stranded DNA binding proteins (SSBs) readily promote replication through hairpins, SSBs are only effective in promoting replication through weak G4s. Using a series of G4s with increasing stabilities, we reveal a threshold above which G4 through-replication is inhibited even with SSBs present, and Pif1 helicase is required. Because Pif1 moves along the template strand with a 5'-3'-directionality, head-on collisions between Pif1 and polymerase δ or γ result in the stimulation of their 3'-exonuclease activity. Both nuclear RPA and mitochondrial SSB play a protective role during DNA replication by preventing excessive DNA degradation caused by the helicase-polymerase conflict.
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http://dx.doi.org/10.1093/nar/gkz608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145523PMC
September 2019

Activation of Tel1 kinase requires Rad50 ATPase and long nucleosome-free DNA but no DNA ends.

J Biol Chem 2019 06 9;294(26):10120-10130. Epub 2019 May 9.

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

In , Tel1 protein kinase, the ortholog of human ataxia telangiectasia-mutated (ATM), is activated in response to DNA double-strand breaks. Biochemical studies with human ATM and genetic studies in yeast suggest that recruitment and activation of Tel1 depends on the heterotrimeric MRX complex, composed of Mre11, Rad50, and Xrs2 (human Nbs1). However, the mechanism of activation of Tel1 by MRX remains unclear, as does the role of effector DNA. Here we demonstrate that dsDNA and MRX activate Tel1 synergistically. Although minimal activation was observed with 80-mer duplex DNA, the optimal effector for Tel1 activation is long, nucleosome-free DNA. However, there is no requirement for DNA double-stranded termini. The ATPase activity of Rad50 is critical for activation. In addition to DNA and Rad50, either Mre11 or Xrs2, but not both, is also required. Each of the three MRX subunits shows a physical association with Tel1. Our study provides a model of how the individual subunits of MRX and DNA regulate Tel1 kinase activity.
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http://dx.doi.org/10.1074/jbc.RA119.008410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664182PMC
June 2019

Solution to the 50-year-old Okazaki-fragment problem.

Authors:
Peter M Burgers

Proc Natl Acad Sci U S A 2019 02 15;116(9):3358-3360. Epub 2019 Feb 15.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110

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http://dx.doi.org/10.1073/pnas.1900372116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397534PMC
February 2019

PCNA accelerates the nucleotide incorporation rate by DNA polymerase δ.

Nucleic Acids Res 2019 02;47(4):1977-1986

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO, USA.

DNA polymerase delta (Pol δ) is responsible for the elongation and maturation of Okazaki fragments in eukaryotic cells. Proliferating cell nuclear antigen (PCNA) recruits Pol δ to the DNA and serves as a processivity factor. Here, we show that PCNA also stimulates the catalytic rate of Saccharomyces cerevisiae Pol δ by >10-fold. We determined template/primer DNA binding affinities and stoichiometries by Pol δ in the absence of PCNA, using electrophoretic mobility shift assays, fluorescence intensity changes and fluorescence anisotropy binding titrations. We provide evidence that Pol δ forms higher ordered complexes upon binding to DNA. The Pol δ catalytic rates in the absence and presence of PCNA were determined at millisecond time resolution using quench flow kinetic measurements. The observed rate for single nucleotide incorporation by a preformed DNA-Pol δ complex in the absence of PCNA was 40 s-1. PCNA enhanced the nucleotide incorporation rate by >10 fold. Compared to wild-type, a growth-defective yeast PCNA mutant (DD41,42AA) showed substantially less stimulation of the Pol δ nucleotide incorporation rate, identifying the face of PCNA that is important for the acceleration of catalysis.
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http://dx.doi.org/10.1093/nar/gky1321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393303PMC
February 2019

Mechanism of Lagging-Strand DNA Replication in Eukaryotes.

Adv Exp Med Biol 2017 ;1042:117-133

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO, USA.

This chapter focuses on the enzymes and mechanisms involved in lagging-strand DNA replication in eukaryotic cells. Recent structural and biochemical progress with DNA polymerase α-primase (Pol α) provides insights how each of the millions of Okazaki fragments in a mammalian cell is primed by the primase subunit and further extended by its polymerase subunit. Rapid kinetic studies of Okazaki fragment elongation by Pol δ illuminate events when the polymerase encounters the double-stranded RNA-DNA block of the preceding Okazaki fragment. This block acts as a progressive molecular break that provides both time and opportunity for the flap endonuclease 1 (FEN1) to access the nascent flap and cut it. The iterative action of Pol δ and FEN1 is coordinated by the replication clamp PCNA and produces a regulated degradation of the RNA primer, thereby preventing the formation of long-strand displacement flaps. Occasional long flaps are further processed by backup nucleases including Dna2.
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http://dx.doi.org/10.1007/978-981-10-6955-0_6DOI Listing
July 2018

A Redox Role for the [4Fe4S] Cluster of Yeast DNA Polymerase δ.

J Am Chem Soc 2017 12 6;139(50):18339-18348. Epub 2017 Dec 6.

Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, California 91125, United States.

A [4Fe4S] cluster in the C-terminal domain of the catalytic subunit of the eukaryotic B-family DNA polymerases is essential for the formation of active multi-subunit complexes. Here we use a combination of electrochemical and biochemical methods to assess the redox activity of the [4Fe4S] cluster in Saccharomyces cerevisiae polymerase (Pol) δ, the lagging strand DNA polymerase. We find that Pol δ bound to DNA is indeed redox-active at physiological potentials, generating a DNA-mediated signal electrochemically with a midpoint potential of 113 ± 5 mV versus NHE. Moreover, biochemical assays following electrochemical oxidation of Pol δ reveal a significant slowing of DNA synthesis that can be fully reversed by reduction of the oxidized form. A similar result is apparent with photooxidation using a DNA-tethered anthraquinone. These results demonstrate that the [4Fe4S] cluster in Pol δ can act as a redox switch for activity, and we propose that this switch can provide a rapid and reversible way to respond to replication stress.
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http://dx.doi.org/10.1021/jacs.7b10284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881389PMC
December 2017

Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication.

Bioessays 2017 08;39(8)

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA.

Biochemical and cryo-electron microscopy studies have just been published revealing interactions among proteins of the yeast replisome that are important for highly coordinated synthesis of the two DNA strands of the nuclear genome. These studies reveal key interactions important for arranging DNA polymerases α, δ, and ϵ for leading and lagging strand replication. The CMG (Mcm2-7, Cdc45, GINS) helicase is central to this interaction network. These are but the latest examples of elegant studies performed in the recent past that lead to a much better understanding of how the eukaryotic replication fork achieves efficient DNA replication that is accurate enough to prevent diseases yet allows evolution.
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http://dx.doi.org/10.1002/bies.201700070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579836PMC
August 2017

Eukaryotic DNA Replication Fork.

Annu Rev Biochem 2017 06 1;86:417-438. Epub 2017 Mar 1.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; email:

This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.
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http://dx.doi.org/10.1146/annurev-biochem-061516-044709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597965PMC
June 2017

Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations.

Nucleic Acids Res 2017 02;45(3):1200-1218

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA.

In yeast, dNTP pools expand drastically during DNA damage response. We show that similar dNTP elevation occurs in strains, in which intrinsic replisome defects promote the participation of error-prone DNA polymerase ζ (Polζ) in replication of undamaged DNA. To understand the significance of dNTP pools increase for Polζ function, we studied the activity and fidelity of four-subunit Polζ (Polζ4) and Polζ4-Rev1 (Polζ5) complexes in vitro at ‘normal S-phase’ and ‘damage-response’ dNTP concentrations. The presence of Rev1 inhibited the activity of Polζ and greatly increased the rate of all three ‘X-dCTP’ mispairs, which Polζ4 alone made extremely inefficiently. Both Polζ4 and Polζ5 were most promiscuous at G nucleotides and frequently generated multiple closely spaced sequence changes. Surprisingly, the shift from ‘S-phase’ to ‘damage-response’ dNTP levels only minimally affected the activity, fidelity and error specificity of Polζ complexes. Moreover, Polζ-dependent mutagenesis triggered by replisome defects or UV irradiation in vivo was not decreased when dNTP synthesis was suppressed by hydroxyurea, indicating that Polζ function does not require high dNTP levels. The results support a model wherein dNTP elevation is needed to facilitate non-mutagenic tolerance pathways, while Polζ synthesis represents a unique mechanism of rescuing stalled replication when dNTP supply is low.
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http://dx.doi.org/10.1093/nar/gkw1149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388397PMC
February 2017

Oxidative DNA damage stalls the human mitochondrial replisome.

Sci Rep 2016 07 1;6:28942. Epub 2016 Jul 1.

Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase γ holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage, and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism.
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http://dx.doi.org/10.1038/srep28942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929447PMC
July 2016

Parallel analysis of ribonucleotide-dependent deletions produced by yeast Top1 in vitro and in vivo.

Nucleic Acids Res 2016 09 1;44(16):7714-21. Epub 2016 Jun 1.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA

Ribonucleotides are the most abundant non-canonical component of yeast genomic DNA and their persistence is associated with a distinctive mutation signature characterized by deletion of a single repeat unit from a short tandem repeat. These deletion events are dependent on DNA topoisomerase I (Top1) and are initiated by Top1 incision at the relevant ribonucleotide 3'-phosphodiester. A requirement for the re-ligation activity of Top1 led us to propose a sequential cleavage model for Top1-dependent mutagenesis at ribonucleotides. Here, we test key features of this model via parallel in vitro and in vivo analyses. We find that the distance between two Top1 cleavage sites determines the deletion size and that this distance is inversely related to the deletion frequency. Following the creation of a gap by two Top1 cleavage events, the tandem repeat provides complementarity that promotes realignment to a nick and subsequent Top1-mediated ligation. Complementarity downstream of the gap promotes deletion formation more effectively than does complementarity upstream of the gap, consistent with constraints to realignment of the strand to which Top1 is covalently bound. Our data fortify sequential Top1 cleavage as the mechanism for ribonucleotide-dependent deletions and provide new insight into the component steps of this process.
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http://dx.doi.org/10.1093/nar/gkw495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027487PMC
September 2016

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

J Biol Chem 2016 Jun 28;291(26):13436-47. Epub 2016 Apr 28.

From the Section of Structural Biology, Department of Medicine, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom and

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.
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http://dx.doi.org/10.1074/jbc.M115.708263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919432PMC
June 2016

Proficient Replication of the Yeast Genome by a Viral DNA Polymerase.

J Biol Chem 2016 May 12;291(22):11698-705. Epub 2016 Apr 12.

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol α, Pol δ, and Pol ϵ. Pol δ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol δ is a three-subunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both iron-sulfur cluster and zinc-binding motifs, which mediate interactions with Pol31, and processive replication with the replication clamp proliferating cell nuclear antigen (PCNA), respectively. We show that the entire N-terminal domain of Pol3, containing polymerase and proofreading activities, could be effectively replaced by those from bacteriophage RB69, and could carry out chromosomal DNA replication in yeast with remarkable high fidelity, provided that adaptive mutations in the replication clamp PCNA were introduced. This result is consistent with the model that all essential interactions for DNA replication in yeast are mediated through the small C-terminal domain of Pol3. The chimeric polymerase carries out processive replication with PCNA in vitro; however, in yeast, it requires an increased involvement of the mutagenic translesion DNA polymerase ζ during DNA replication.
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http://dx.doi.org/10.1074/jbc.M116.728741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882438PMC
May 2016

Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale.

Nat Struct Mol Biol 2016 05 11;23(5):402-8. Epub 2016 Apr 11.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri, USA.

DNA polymerase delta (Pol δ) is responsible for elongation and maturation of Okazaki fragments. Pol δ and the flap endonuclease FEN1, coordinated by the PCNA clamp, remove RNA primers and produce ligatable nicks. We studied this process in the Saccharomyces cerevisiae machinery at millisecond resolution. During elongation, PCNA increased the Pol δ catalytic rate by >30-fold. When Pol δ invaded double-stranded RNA-DNA representing unmatured Okazaki fragments, the incorporation rate of each nucleotide decreased successively to 10-20% that of the preceding nucleotide. Thus, the nascent flap acts as a progressive molecular brake on the polymerase, and consequently FEN1 cuts predominantly single-nucleotide flaps. Kinetic and enzyme-trapping experiments support a model in which a stable PCNA-DNA-Pol δ-FEN1 complex moves processively through iterative steps of nick translation, ultimately completely removing primer RNA. Finally, whereas elongation rates are under dynamic dNTP control, maturation rates are buffered against changes in dNTP concentrations.
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http://dx.doi.org/10.1038/nsmb.3207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857878PMC
May 2016

Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ.

Nucleic Acids Res 2016 05 21;44(8):3811-9. Epub 2016 Mar 21.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA

Using an in vitro reconstituted system in this work we provide direct evidence that the yeast repressor/activator protein 1 (Rap1), tightly bound to its consensus site, forms a strong non-polar barrier for the strand displacement activity of DNA polymerase δ. We propose that relief of inhibition may be mediated by the activity of an accessory helicase. To this end, we show that Pif1, a 5'-3' helicase, not only stimulates the strand displacement activity of Pol δ but it also allows efficient replication through the block, by removing bound Rap1 in front of the polymerase. This stimulatory activity of Pif1 is not limited to the displacement of a single Rap1 molecule; Pif1 also allows Pol δ to carry out DNA synthesis across an array of bound Rap1 molecules that mimics a telomeric DNA-protein assembly. This activity of Pif1 represents a novel function of this helicase during DNA replication.
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http://dx.doi.org/10.1093/nar/gkw181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856994PMC
May 2016

Who Is Leading the Replication Fork, Pol ε or Pol δ?

Mol Cell 2016 Feb;61(4):492-493

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA. Electronic address:

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http://dx.doi.org/10.1016/j.molcel.2016.01.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838066PMC
February 2016

Probing the Mec1ATR Checkpoint Activation Mechanism with Small Peptides.

J Biol Chem 2016 Jan 23;291(1):393-401. Epub 2015 Oct 23.

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110,

Yeast Mec1, the ortholog of human ATR, is the apical protein kinase that initiates the cell cycle checkpoint in response to DNA damage and replication stress. The basal activity of Mec1 kinase is activated by cell cycle phase-specific activators. Three distinct activators stimulate Mec1 kinase using an intrinsically disordered domain of the protein. These are the Ddc1 subunit of the 9-1-1 checkpoint clamp (ortholog of human and Schizosaccharomyces pombe Rad9), the replication initiator Dpb11 (ortholog of human TopBP1 and S. pombe Cut5), and the multifunctional nuclease/helicase Dna2. Here, we use small peptides to determine the requirements for Mec1 activation. For Ddc1, we identify two essential aromatic amino acids in a hydrophobic environment that when fused together are proficient activators. Using this increased insight, we have been able to identify homologous motifs in S. pombe Rad9 that can activate Mec1. Furthermore, we show that a 9-amino acid Dna2-based peptide is sufficient for Mec1 activation. Studies with mutant activators suggest that binding of an activator to Mec1 is a two-step process, the first step involving the obligatory binding of essential aromatic amino acids to Mec1, followed by an enhancement in binding energy through interactions with neighboring sequences.
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http://dx.doi.org/10.1074/jbc.M115.687145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4697174PMC
January 2016

Yet another job for Dna2: Checkpoint activation.

DNA Repair (Amst) 2015 Aug 1;32:17-23. Epub 2015 May 1.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Mec1 (ATR in humans) is the principal kinase responsible for checkpoint activation in response to replication stress and DNA damage in Saccharomyces cerevisiae. Checkpoint initiation requires stimulation of Mec1 kinase activity by specific activators. The complexity of checkpoint initiation in yeast increases with the complexity of chromosomal states during the different phases of the cell cycle. In G1 phase, the checkpoint clamp 9-1-1 is both necessary and sufficient for full activation of Mec1 kinase whereas in G2/M, robust checkpoint function requires both 9-1-1 and the replisome assembly protein Dpb11 (human TopBP1). A third activator, Dna2, is employed specifically during S phase to stimulate Mec1 kinase and to initiate the replication checkpoint. Dna2 is an essential nuclease-helicase that is required for proper Okazaki fragment maturation, for double-strand break repair, and for protecting stalled replication forks. Remarkably, all three Mec1 activators use an unstructured region of the protein, containing two critically important aromatic residues, in order to activate Mec1. A role for these checkpoint activators in channeling aberrant replication structures into checkpoint complexes is discussed.
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http://dx.doi.org/10.1016/j.dnarep.2015.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522331PMC
August 2015

Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5'-flaps.

Nucleic Acids Res 2015 Apr 26;43(8):4179-90. Epub 2015 Mar 26.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA

The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3'-5' exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo(-) to carry out strand displacement synthesis and discovered that it is regulated by the 5'-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5'-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5'-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities.
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http://dx.doi.org/10.1093/nar/gkv260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417170PMC
April 2015

Error-free and mutagenic processing of topoisomerase 1-provoked damage at genomic ribonucleotides.

EMBO J 2015 May 16;34(9):1259-69. Epub 2015 Mar 16.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA

Genomic ribonucleotides incorporated during DNA replication are commonly repaired by RNase H2-dependent ribonucleotide excision repair (RER). When RNase H2 is compromised, such as in Aicardi-Goutières patients, genomic ribonucleotides either persist or are processed by DNA topoisomerase 1 (Top1) by either error-free or mutagenic repair. Here, we present a biochemical analysis of these pathways. Top1 cleavage at genomic ribonucleotides can produce ribonucleoside-2',3'-cyclic phosphate-terminated nicks. Remarkably, this nick is rapidly reverted by Top1, thereby providing another opportunity for repair by RER. However, the 2',3'-cyclic phosphate-terminated nick is also processed by Top1 incision, generally 2 nucleotides upstream of the nick, which produces a covalent Top1-DNA complex with a 2-nucleotide gap. We show that these covalent complexes can be processed by proteolysis, followed by removal of the phospho-peptide by Tdp1 and the 3'-phosphate by Tpp1 to mediate error-free repair. However, when the 2-nucleotide gap is associated with a dinucleotide repeat sequence, sequence slippage re-alignment followed by Top1-mediated religation can occur which results in 2-nucleotide deletion. The efficiency of deletion formation shows strong sequence-context dependence.
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http://dx.doi.org/10.15252/embj.201490868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426484PMC
May 2015

Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific.

Nat Struct Mol Biol 2015 Apr 9;22(4):291-7. Epub 2015 Mar 9.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA.

Ribonucleotides incorporated during DNA replication are removed by RNase H2-dependent ribonucleotide excision repair (RER). In RER-defective yeast, topoisomerase 1 (Top1) incises DNA at unrepaired ribonucleotides, initiating their removal, but this is accompanied by RNA-DNA-damage phenotypes. Here we show that these phenotypes are incurred by a high level of ribonucleotides incorporated by a leading strand-replicase variant, DNA polymerase (Pol) ɛ, but not by orthologous variants of the lagging-strand replicases, Pols α or δ. Moreover, loss of both RNases H1 and H2 is lethal in combination with increased ribonucleotide incorporation by Pol ɛ but not by Pols α or δ. Several explanations for this asymmetry are considered, including the idea that Top1 incision at ribonucleotides relieves torsional stress in the nascent leading strand but not in the nascent lagging strand, in which preexisting nicks prevent the accumulation of superhelical tension.
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http://dx.doi.org/10.1038/nsmb.2989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4835660PMC
April 2015

Eukaryotic DNA polymerase ζ.

DNA Repair (Amst) 2015 May 19;29:47-55. Epub 2015 Feb 19.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

This review focuses on eukaryotic DNA polymerase ζ (Pol ζ), the enzyme responsible for the bulk of mutagenesis in eukaryotic cells in response to DNA damage. Pol ζ is also responsible for a large portion of mutagenesis during normal cell growth, in response to spontaneous damage or to certain DNA structures and other blocks that stall DNA replication forks. Novel insights in mutagenesis have been derived from recent advances in the elucidation of the subunit structure of Pol ζ. The lagging strand DNA polymerase δ shares the small Pol31 and Pol32 subunits with the Rev3-Rev7 core assembly giving a four subunit Pol ζ complex that is the active form in mutagenesis. Furthermore, Pol ζ forms essential interactions with the mutasome assembly factor Rev1 and with proliferating cell nuclear antigen (PCNA). These interactions are modulated by posttranslational modifications such as ubiquitination and phosphorylation that enhance translesion synthesis (TLS) and mutagenesis.
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http://dx.doi.org/10.1016/j.dnarep.2015.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426032PMC
May 2015
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