Abstract
Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches upon exposure of cells to DNA damage induced either by MMS alkylation or by UV-radiation. We have also found that the potential Mec1/Rad53 balance modulation is independent from Rad6/Rad18-mediated PCNA ubiquitylation, as mec1Δ or rad53Δ mutants show no defects in the modification of the sliding clamp, therefore, we infer that it is likely exerted by acting on TLS polymerases and/or template switching targets.
Highlights
The DNA damage tolerance mechanism allows DNA replication forks to progress through chemically altered, or damaged, template strands preventing irreversible fork collapse during S phase
Rev1 is a deoxycytidyltransferase involved in the bypass of abasic sites in damaged DNA and it forms a complex with the subunits of DNA polymerase zeta (f) Rev3p and Rev7p, which are involved in error-prone lesion bypass (TLS DNA polymerases) [20,21] (Figure S1)
We carried out ten-fold serial dilutions and found that mms2D rad53Ha cells were less sensitive to the chronic presence of methyl methanesulfonate (MMS) than mms2D single mutants (Fig. 1B), suggesting that resistance to DNA damage in rad53Ha does not depends on the error-free branch of lesion bypass
Summary
The DNA damage tolerance mechanism allows DNA replication forks to progress through chemically altered, or damaged, template strands preventing irreversible fork collapse during S phase. The balance between error-prone and error-free TLS mechanisms is different between different species of living organisms, or even in distinct types of cells within the same organism (for a review, see [2]). This distinctive regulation may reflect changes in TLS polymerase usage in different cells or in dealing with different kinds of DNA lesions [2,3]. These changes in the relative balance suggest the existence of a modulatory mechanism of control. In the unicellular budding yeast, S. cerevisiae, it is thought that error-prone and error-free branches are unbalanced towards the latter, as reported for the replication of plasmids with a defined photoproduct [4], such that cells bypass DNA lesions without necessarily increasing the mutagenic rate
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