HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells

Vipul Shukla; Levon Halabelian; Sanjana Balagere; Daniela Samaniego-Castruita; Douglas E. Feldman; Cheryl H. Arrowsmith; Anjana Rao; L. Aravind

doi:10.1016/j.molcel.2019.10.031

HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells

Vipul Shukla, Levon Halabelian, Sanjana Balagere, Daniela Samaniego-Castruita, Douglas E. Feldman, Cheryl H. Arrowsmith^*, Anjana Rao, L. Aravind

^*Corresponding author for this work

Cell & Developmental Biology

Research output: Contribution to journal › Article › peer-review

34 Scopus citations

Abstract

HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.

Original language	English (US)
Pages (from-to)	384-394.e4
Journal	Molecular cell
Volume	77
Issue number	2
DOIs	https://doi.org/10.1016/j.molcel.2019.10.031
State	Published - Jan 16 2020

Funding

We would like to thank Dr. J. Stark for providing the c-NHEJ and Alt-EJ U2OS reporter cell lines and C. Kim, L. Nosworthy, D. Hinz, and C. Dillingham (LJI Flow Cytometry Core) for help with cell sorting. BD FACSAria II is supported by NIH ( NIH S10OD016262 , NIH S10RR027366 ). We thank Dr. H. Wyatt for the recommendations on the exonuclease protection assay. This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract DE-AC02-05CH11231. V.S. is supported by Leukemia and Lymphoma Society Postdoctoral Fellowship ( 5463-18 ). D.S.-C. is supported by CONACYT/UCMEXUS Fellowship . The Structural Genomics Consortium is a registered charity (1097737) that receives funds from AbbVie , Bayer Pharma AG , Boehringer Ingelheim (Vienna, Austria) , Canada Foundation for Innovation , Eshelman Institute for Innovation , Genome Canada through Ontario Genomics Institute ( OGI-055 ), Innovative Medicines Initiative (EU/EFPIA) ( ULTRA-DD: 115766 ), Janssen, Merck & Co. , Novartis Pharma AG , Ontario Ministry of Research Innovation and Science (MRIS), Pfizer, São Paulo Research Foundation -FAPESP, Takeda , and the Wellcome Trust . This work is supported by the Canadian Institutes of Health Research ( FDN154328 to C.H.A.), Natural Sciences and Engineering Research Council ( RGPIN-2015-05939 to C.H.A.), NIH ( R35 CA210043 and R01 AI109842 to A.R.), and intramural funds of the National Library of Medicine, NIH, USA (to L.A.). We would like to thank Dr. J. Stark for providing the c-NHEJ and Alt-EJ U2OS reporter cell lines and C. Kim, L. Nosworthy, D. Hinz, and C. Dillingham (LJI Flow Cytometry Core) for help with cell sorting. BD FACSAria II is supported by NIH (NIH S10OD016262, NIH S10RR027366). We thank Dr. H. Wyatt for the recommendations on the exonuclease protection assay. This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract DE-AC02-05CH11231. V.S. is supported by Leukemia and Lymphoma Society Postdoctoral Fellowship (5463-18). D.S.-C. is supported by CONACYT/UCMEXUS Fellowship. The Structural Genomics Consortium is a registered charity (1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim (Vienna, Austria), Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute (OGI-055), Innovative Medicines Initiative (EU/EFPIA) (ULTRA-DD: 115766), Janssen, Merck & Co., Novartis Pharma AG, Ontario Ministry of Research Innovation and Science (MRIS), Pfizer, S?o Paulo Research Foundation-FAPESP, Takeda, and the Wellcome Trust. This work is supported by the Canadian Institutes of Health Research (FDN154328 to C.H.A.), Natural Sciences and Engineering Research Council (RGPIN-2015-05939 to C.H.A.), NIH (R35 CA210043 and R01 AI109842 to A.R.), and intramural funds of the National Library of Medicine, NIH, USA (to L.A.). V.S. conceptualized the experiments, acquired and analyzed the data, and wrote the manuscript. L.H. performed structural studies of the HMCES protein, drafted parts of the manuscript, helped with the interpretation of data, and reviewed the manuscript. S.B. performed cloning of the HMCES variants. D.S.-C. performed switching experiments in primary B cells. D.E.F. provided the Hmces-deficient mice used in the study. L.A. contributed to conceptualization of the experiments, discussion of the data, and review of the manuscript. C.H.A. supervised the structural studies of HMCES, interpretation of the data, and review of the manuscript. A.R. supervised the studies, conceptualized the experiments, helped with the interpretation of data, and wrote the manuscript. The authors declare no competing interests.

Keywords

DNA double-strand break (DNA DSB) repair
HMCES
TET proteins
alternative end joining
class switch recombination (CSR)
oxidized methylcytosines

ASJC Scopus subject areas

Molecular Biology
Cell Biology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.molcel.2019.10.031

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title = "HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells",

abstract = "HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.",

keywords = "DNA double-strand break (DNA DSB) repair, HMCES, TET proteins, alternative end joining, class switch recombination (CSR), oxidized methylcytosines",

author = "Vipul Shukla and Levon Halabelian and Sanjana Balagere and Daniela Samaniego-Castruita and Feldman, {Douglas E.} and Arrowsmith, {Cheryl H.} and Anjana Rao and L. Aravind",

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AU - Shukla, Vipul

AU - Halabelian, Levon

AU - Balagere, Sanjana

AU - Samaniego-Castruita, Daniela

AU - Feldman, Douglas E.

AU - Arrowsmith, Cheryl H.

AU - Rao, Anjana

AU - Aravind, L.

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KW - oxidized methylcytosines

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HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells

Abstract

Funding

Keywords

ASJC Scopus subject areas

UN SDGs

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