Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jun 2;31(9):107705.
doi: 10.1016/j.celrep.2020.107705.

HMCES Maintains Replication Fork Progression and Prevents Double-Strand Breaks in Response to APOBEC Deamination and Abasic Site Formation

Kavi P M Mehta  1 , Courtney A Lovejoy  1 , Runxiang Zhao  1 , Darren R Heintzman  1 , David Cortez  2
Affiliations

HMCES Maintains Replication Fork Progression and Prevents Double-Strand Breaks in Response to APOBEC Deamination and Abasic Site Formation

Kavi P M Mehta et al. Cell Rep. .

Abstract

5-Hydroxymethylcytosine (5hmC) binding, ES-cell-specific (HMCES) crosslinks to apurinic or apyrimidinic (AP, abasic) sites in single-strand DNA (ssDNA). To determine whether HMCES responds to the ssDNA abasic site in cells, we exploited the activity of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3A (APOBEC3A). APOBEC3A preferentially deaminates cytosines to uracils in ssDNA, which are then converted to abasic sites by uracil DNA glycosylase. We find that HMCES-deficient cells are hypersensitive to nuclear APOBEC3A localization. HMCES relocalizes to chromatin in response to nuclear APOBEC3A and protects abasic sites from processing into double-strand breaks (DSBs). Abasic sites induced by APOBEC3A slow both leading and lagging strand synthesis, and HMCES prevents further slowing of the replication fork by translesion synthesis (TLS) polymerases zeta (Polζ) and kappa (Polκ). Thus, our study provides direct evidence that HMCES responds to ssDNA abasic sites in cells to prevent DNA cleavage and balance the engagement of TLS polymerases.

Keywords: APOBEC; DNA damage; DNA repair; HMCES; abasic site; damage tolerance; replication stress; translesion synthesis.

PubMed Disclaimer

Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. HMCES-Deficient Cells Are Hypersensitive to Nuclear APOBEC3A Expression, Which Promotes HMCES Localization to the Chromatin
(A) Representative images of a colony-viability assay in hTERT-RPE-1 GFP-APOBEC3A-ERT2 cells transfected with non-targeting control (NT) or HMCES (H-1 and H-2) siRNAs and treated with 4-OHT for 24 h. (B and C) Quantitation of clonogenic survival assays for RPE A3A (B) and HCT116 A3A (C). Means and SD of 3 experiments. p values were derived from an ANOVA with Holm-Sidak post-test. (D and E) Quantitation of the insoluble HMCES PLA signal after 4-OHT treatment in EdU-positive (D) and EdU-negative (E) hTERT-RPE-1 cells, with and without stable expression of GFP-APOBEC3A-ERT2. (−1°, no primary HMCES antibody). Each data point represents the HMCES-PLA-integrated nuclear intensity in one cell. Bars represent the median, and p values were derived from an ANOVA with Dunn’s multiple comparisons post-test. See also Figures S1 and S2.
Figure 2.
Figure 2.. HMCES Protects Cells from APOBEC3A-Induced DSBs
(A)Cells were transfected with HMCESsiRNAs (H-1, H-2) or a non-targetingsiRNA (NT) and treated with 4-OHT for 1 h as indicated prior to neutral comet assay. (B) Cells were transfected with siRNA and treated with 4-OHT. The agarose plugs in the neutral comet assay were treated with purified APE-I for the indicated samples. All bars represent the median, and p values were derived from an ANOVA with Dunn’s multiple comparisons post-test. See also Figure S3.
Figure 3.
Figure 3.. APOBEC3A-Induced Abasic Sites Slow Replication Elongation
(A) Three clones (c2, c9, and c11) of HCT116 GFP-APOBEC3A-ERT2 cells, or parental HCT116 cells, were incubated with 4-OHT, CldU, and IdU, as indicated, and processed for DNA combing. (B) The uracil-DNA glycosylase inhibitor (UGI) or an empty vector (EV) was introduced by lentiviral infection before DNA combing. (C) hTERT-RPE-1 cells were harvested and lysed 16 h after UGI was introduced by lentiviral infection to assess UNG inhibition. (D) hTERT-RPE-1 GFP-APOBEC3A-ERT2 cells were infected with EV or UGI before DNA combing. All bars represent the median, and p values were derived from an ANOVA with Dunn’s multiple comparisons post-test.
Figure 4.
Figure 4.. HMCES and TLS Slow Replication Elongation in Response to Nuclear APOBEC3A
(A) Cells were transfected with HMCES (H-1 and H-2), POLK, or a non-targeting siRNA (NT) and were incubated with 4-OHT before DNA combing. (B) UGI or empty vector (EV) was introduced by lentiviral infection. (C) Cells were transfected with HMCES or non-targeting siRNA, treated with DMSO, 4-OHT, and 10 μM JH-RE-06 for 30 min as indicated, and processed for DNA combing. (D) Cells were transfected with HMCES and POLK siRNA, treated with 4-OHT, and processed for DNA combing. Bars represent the median, and p values were derived from an ANOVA with Dunn’s multiple comparisons post-test.
See this image and copyright information in PMC

References

    1. Aravind L, Anand S, and Iyer LM (2013). Novel autoproteolytic and DNA-damage sensing components in the bacterial SOS response and oxidized methylcytosine-induced eukaryotic DNA demethylation systems. Biol. Direct 8, 20. - PMC - PubMed
    1. Bennett SE, Schimerlik MI, and Mosbaugh DW (1993). Kinetics of the uracil-DNA glycosylase/inhibitor protein association. Ung interaction with Ugi, nucleic acids, and uracil compounds. J. Biol. Chem 268, 26879–26885. - PubMed
    1. Boiteux S, and Guillet M (2004). Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae. DNA Repair (Amst.) 3, 1–12. - PubMed
    1. Burns MB, Lackey L, Carpenter MA, Rathore A, Land AM, Leonard B, Refsland EW, Kotandeniya D, Tretyakova N, Nikas JB, et al. (2013). APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494, 366–370. - PMC - PubMed
    1. Choi JY, Lim S, Kim EJ, Jo A, and Guengerich FP (2010). Translesion synthesis across abasic lesions by human B-family and Y-family DNA polymerases α, δ, η, ι, κ, and REV1. J. Mol. Biol 404, 34–44. - PMC - PubMed

Publication types

MeSH terms