TY - JOUR
T1 - Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites
AU - Twayana, Shyam
AU - Bacolla, Albino
AU - Barreto-Galvez, Angelica
AU - De-Paula, Ruth B.
AU - Drosopoulos, William C.
AU - Kosiyatrakul, Settapong T.
AU - Bouhassira, Eric E.
AU - Tainer, John A.
AU - Madireddy, Advaitha
AU - Schildkraut, Carl L.
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank Kristin A. Eckert for XPV-F Pol eta−/− and XPV-F + Pol eta fibroblasts and Katharina Schlacher for helpful discussions. This work was supported in part by NIH Grants 5R01-GM045751 and R01-CA085344 (to C.L.S.); National Cancer Institute Cancer Center Support Grant P30-CA013330 for use of a core facility at Einstein; NIH Grants R00-HL136870-05 and R00-HL136870-04S1 (to A.M.); NIH Grants CA220430 and CA092584 (to J.A.T.); and a Robert A. Welch Chair in Chemistry (J.A.T.). S.T. was supported by NIH Training Grant T-32 NIH 5T32AG023475. The research used the Bridges/ Bridges2 Pittsburgh Supercomputing Center through the Extreme Science and Engineering Discovery Environment, which is supported by NSF Grants ACI-1445606 and ACI-1548562.
Publisher Copyright:
© 2021 National Academy of Sciences. All rights reserved.
PY - 2021/11/30
Y1 - 2021/11/30
N2 - Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFSFRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.
AB - Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFSFRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.
KW - Common fragile sites
KW - Non-B DNA
KW - Polymerase eta
KW - Replication fork pause
KW - SNP
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U2 - 10.1073/pnas.2106477118
DO - 10.1073/pnas.2106477118
M3 - Article
C2 - 34815340
AN - SCOPUS:85120302737
SN - 0027-8424
VL - 118
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 48
M1 - e2106477118
ER -