Publications by authors named "Renjing Wang"

8 Publications

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DNA clamp function of the monoubiquitinated Fanconi anaemia ID complex.

Nature 2020 04 11;580(7802):278-282. Epub 2020 Mar 11.

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

The ID complex, involving the proteins FANCI and FANCD2, is required for the repair of DNA interstrand crosslinks (ICL) and related lesions. These proteins are mutated in Fanconi anaemia, a disease in which patients are predisposed to cancer. The Fanconi anaemia pathway of ICL repair is activated when a replication fork stalls at an ICL; this triggers monoubiquitination of the ID complex, in which one ubiquitin molecule is conjugated to each of FANCI and FANCD2. Monoubiquitination of ID is essential for ICL repair by excision, translesion synthesis and homologous recombination; however, its function remains unknown. Here we report a cryo-electron microscopy structure of the monoubiquitinated human ID complex bound to DNA, and reveal that it forms a closed ring that encircles the DNA. By comparison with the structure of the non-ubiquitinated ID complex bound to ICL DNA-which we also report here-we show that monoubiquitination triggers a complete rearrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. These structures-together with biochemical data-indicate that the monoubiquitinated ID complex loses its preference for ICL and related branched DNA structures, and becomes a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how monoubiquitination in general can induce an alternative protein structure with a new function.
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http://dx.doi.org/10.1038/s41586-020-2110-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398534PMC
April 2020

DNA repair. Mechanism of DNA interstrand cross-link processing by repair nuclease FAN1.

Science 2014 Nov;346(6213):1127-30

Structural Biology Program and Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

DNA interstrand cross-links (ICLs) are highly toxic lesions associated with cancer and degenerative diseases. ICLs can be repaired by the Fanconi anemia (FA) pathway and through FA-independent processes involving the FAN1 nuclease. In this work, FAN1-DNA crystal structures and biochemical data reveal that human FAN1 cleaves DNA successively at every third nucleotide. In vitro, this exonuclease mechanism allows FAN1 to excise an ICL from one strand through flanking incisions. DNA access requires a 5'-terminal phosphate anchor at a nick or a 1- or 2-nucleotide flap and is augmented by a 3' flap, suggesting that FAN1 action is coupled to DNA synthesis or recombination. FAN1's mechanism of ICL excision is well suited for processing other localized DNA adducts as well.
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http://dx.doi.org/10.1126/science.1258973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285437PMC
November 2014

Structure of the polycomb group protein PCGF1 in complex with BCOR reveals basis for binding selectivity of PCGF homologs.

Structure 2013 Apr 21;21(4):665-71. Epub 2013 Mar 21.

Department of Biochemistry and CTRC, University of Texas Health Science Center at San Antonio, MSC 7760, 7703 Floyd Curl Drive, San Antonio, TX 78229-3990, USA.

Polycomb-group RING finger homologs (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, and PCGF6) are critical components in the assembly of distinct Polycomb repression complex 1 (PRC1)-related complexes. Here, we identify a protein interaction domain in BCL6 corepressor, BCOR, which binds the RING finger- and WD40-associated ubiquitin-like (RAWUL) domain of PCGF1 (NSPC1) and PCGF3 but not of PCGF2 (MEL18) or PCGF4 (BMI1). Because of the selective binding, we have named this domain PCGF Ub-like fold discriminator (PUFD). The structure of BCOR PUFD bound to PCGF1 reveals that (1) PUFD binds to the same surfaces as observed for a different Polycomb group RAWUL domain and (2) the ability of PUFD to discriminate among RAWULs stems from the identity of specific residues within these interaction surfaces. These data show the molecular basis for determining the binding preference for a PCGF homolog, which ultimately helps determine the identity of the larger PRC1-like assembly.
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http://dx.doi.org/10.1016/j.str.2013.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4003909PMC
April 2013

The growth-suppressive function of the polycomb group protein polyhomeotic is mediated by polymerization of its sterile alpha motif (SAM) domain.

J Biol Chem 2012 Mar 24;287(12):8702-13. Epub 2012 Jan 24.

Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3990, USA.

Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.
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http://dx.doi.org/10.1074/jbc.M111.336115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308824PMC
March 2012

Identification of nucleic acid binding residues in the FCS domain of the polycomb group protein polyhomeotic.

Biochemistry 2011 Jun 12;50(22):4998-5007. Epub 2011 May 12.

Department of Biochemistry, University of Texas Health Science Center at San Antonio, MSC 7760, San Antonio, Texas 78229-3990, United States.

Polycomb group (PcG) proteins maintain the silent state of developmentally important genes. Recent evidence indicates that noncoding RNAs also play an important role in targeting PcG proteins to chromatin and PcG-mediated chromatin organization, although the molecular basis for how PcG and RNA function in concert remains unclear. The Phe-Cys-Ser (FCS) domain, named for three consecutive residues conserved in this domain, is a 30-40-residue Zn(2+) binding motif found in a number of PcG proteins. The FCS domain has been shown to bind RNA in a non-sequence specific manner, but how it does so is not known. Here, we present the three-dimensional structure of the FCS domain from human Polyhomeotic homologue 1 (HPH1, also known as PHC1) determined using multidimensional nuclear magnetic resonance methods. Chemical shift perturbations upon addition of RNA and DNA resulted in the identification of Lys 816 as a potentially important residue required for nucleic acid binding. The role played by this residue in Polyhomeotic function was demonstrated in a transcription assay conducted in Drosophila S2 cells. Mutation of the Arg residue to Ala in the Drosophila Polyhomeotic (Ph) protein, which is equivalent to Lys 816 in HPH1, was unable to repress transcription of a reporter gene to the level of wild-type Ph. These results suggest that direct interaction between the Ph FCS domain and nucleic acids is required for Ph-mediated repression.
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http://dx.doi.org/10.1021/bi101487sDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938326PMC
June 2011

Polycomb group targeting through different binding partners of RING1B C-terminal domain.

Structure 2010 Aug;18(8):966-75

Department of Biochemistry, University of Texas Health Science Center at San Antonio, MSC 7760, 7703 Floyd Curl Drive, San Antonio, TX 78229-3990, USA.

RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure.
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http://dx.doi.org/10.1016/j.str.2010.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445678PMC
August 2010

Characterization of reversible associations by sedimentation velocity with UltraScan.

Macromol Biosci 2010 Jul;10(7):775-82

Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.

We compare here the utility of sedimentation velocity (SV) to sedimentation equilibrium (SE) analysis for the characterization of reversible systems. Genetic algorithm optimization in UltraScan is used to optimize the model and to obtain solution properties of all components present in the system. We apply our method to synthetic and experimental data, and suggest limits for the accessible kinetic range. We conclude that equilibrium constants obtained from SV and SE analysis are equivalent, but that SV experiments provide better confidence for the K(d), can better account for the presence of contaminants and provide additional information including rate constants and shape parameters.
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http://dx.doi.org/10.1002/mabi.200900481DOI Listing
July 2010

Structural transitions of the RING1B C-terminal region upon binding the polycomb cbox domain.

Biochemistry 2008 Aug 11;47(31):8007-15. Epub 2008 Jul 11.

Department of Biochemistry, University of Texas Health Science Center at San Antonio, MSC 7760, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900, USA.

Polycomb group (PcG) proteins are required for maintaining cell identity and stem cell self-renewal. RING1B and Polycomb (Pc) are two components of a multiprotein complex called polycomb repression complex 1 (PRC1) that is essential for establishing and maintaining long-term repressed gene states. Here we characterize the interaction between the C-terminal region of RING1B (C-RING1B) and the Pc cbox domain. The C-RING1B-cbox interaction displays a 1:1 stoichiometry with dissociation constants ranging from 9.2 to 180 nM for the different Pc orthologues. NMR analysis of C-RING1B alone reveals line broadening. However, when it is in complex with the cbox domain, there is a striking change to the NMR spectrum indicative of conformational tightening. This conformational change may arise from the organization of the C-RING1B subdomains. The C-terminal regions of all PcG RING1 proteins are composed of two stretches of conserved sequences separated by a variable linker sequence. While the entire C-RING1B region is required for cbox binding, the N- and C-terminal halves of C-RING1B can be separated and are able to interact, suggesting the presence of an intramolecular interaction within C-RING1B. The flexibility within the C-RING1B structure allowing transitions between the intramolecular bound and unbound states may cause the broadened peaks of the C-RING1B NMR spectrum. Binding the cbox domain stabilizes C-RING1B, whereby broadening is eliminated. The presence of flexible regions could allow C-RING1B to bind a variety of different factors, ultimately recruiting RING1B and its associated PcG proteins to different genomic loci.
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http://dx.doi.org/10.1021/bi800857fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442619PMC
August 2008