A novel mechanism for the inhibition of DNA synthesis following methylation: The effect of N-methyl-N-nitrosourea on HeLa cells

High Doses of SN1 Type Methylating Agents Activate DNA Damage Signaling Cascades that are Largely Independent of Mismatch Repair

“…10 The arrest was accompanied by the post-translational modification of several downstream targets of the ATR/CHK1 kinases, as well as by the formation of nuclear foci of the single-strand DNA binding protein RPA, the phosphorylated form of histone H2AX (γ-H2AX) and polypeptide(s) modified in the SQ/TQ motifs. The formation of RPA foci appeared to support the model proposed by Plant and Roberts in 1971, 11 which suggested that during the first S-phase, replication past O 6 -methylguanine residues leaves gaps in the newly-synthesized strand that are converted to double-strand breaks (DSBs) in the second S-phase. While trying to further elucidate the molecular transactions that bring about O 6 -methylguanine-associated cell death, we carried out dose response experiments in our cell system.…”

Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA

“…In this scenario, iterative attempts of the replicative polymerase to find a perfect match for Me G would be expected to stall, or at least slow down, the progression of the replication fork. DNA synthesis across Me G has indeed been shown to retard replicative polymerases in vitro (Haracska et al 2000;York and Modrich 2006); however, neither the early experiments (Plant and Roberts 1971) nor our measurements of gross replication rates in mammalian cells (Fig. 2), nor examination of the progression of replication forks from a defined origin in yeast cells ( Supplementary Fig.…”

Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase

“…More than 30 years ago, Plant and Roberts (1971) suggested that replication past 6Me G in the template strand during the first S phase may give rise to single-stranded gaps, which are converted into DSBs during the second replication cycle. This was long thought to be unlikely, as DNA polymerases were expected to repair gaps remaining from incomplete replication during the G 2 phase.…”

“…Second, assuming that the single-stranded gaps do indeed form, how could they persist until the subsequent S phase as suggested (Plant and Roberts 1971;Kaina et al 1997)? This could be the result of a combination of factors.…”