Abstract
DNA crosslinks can block replication in vitro and slow down S phase progression in vivo. We characterized the effect of mitomycin C crosslinker on S phase globally and on individual replication forks in wild type and FANCD2-deficient human cells. FANCD2 is critical to crosslink repair, and is also implicated in facilitating DNA replication. We used DNA fiber analysis to demonstrate persistent reduction in abundance but not progression rate of replication forks during an S phase of MMC-treated cells. FANCD2 deficiency did not eliminate this phenotype. Immunoprecipitation of EdU-labeled DNA indicated that replication was not suppressed in the domains that were undergoing response to MMC as marked by the presence of γH2AX, and in fact γH2AX was overrepresented on DNA that had replicated immediately after MMC in wild type through less so in FANCD2-depleted cells. FANCD2-depleted cells also produced fewer tracks of uninterrupted replication of up to 240Kb long, regardless of MMC treatment. Overall, the data suggest that crosslinks may not pose a block to S phase as a whole, but instead profoundly change its progress by reducing density of replication forks and causing at least a fraction of forks to operate within a DNA damage response-altered chromatin.
Highlights
Cellular response to DNA crosslinks involves more than one DNA repair and checkpoint pathway, making these lesions a likely challenge to cancer cells that have either lost or deregulated at least some of the DNA damage response machineries [1]
Accumulation of γH2AX was evident in mitomycin C (MMC)-treated S phase (EdU+) and non-S phase (EdU-) primary cells for up to 9 hrs after the drug (Figures 1E, S3A, B)
Transient inhibition of DNA replication after DNA damage in eukaryotic cells has been known for years, it mechanistic dissection continues to reveal layers of complexity
Summary
Cellular response to DNA crosslinks involves more than one DNA repair and checkpoint pathway, making these lesions a likely challenge to cancer cells that have either lost or deregulated at least some of the DNA damage response machineries [1]. Crosslinker drugs such as CisPt, nitrogen mustards, mitomycin C (MMC) and others, generate a mixture of intra- and interstrand links as DNA adducts, which are capable of blocking DNA polymerases in a test tube. As with other kinds of damage, genomic replication as a whole may be altered by crosslinks rather than blocked, through a combination of “filling in” around lesions or bypassing them
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