Abstract
In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5′ to 3′ end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5′ to 3′ resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells.
Highlights
DNA double strand breaks (DSBs) are highly deleterious events that may lead to chromosomal abnormalities, cell death and cancer
We find that ␥ -H2AX is retained around a DSB after caffeine treatment indicating that the modification is stable, and does not depend on continuous Mec1/Tel1 activity
Western blotting in S. cerevisiae was carried out using the trichloroacetic acid (TCA) protocol described by Pellicioli et al [55]. ␣-Rho1 antibody was a generous gift from Satoshi Yoshida (Brandeis, MA, USA). ␣-GFP, ␣CMyc and ␣-PGK1 antibodies were obtained from Abcam. ␣-V5 antibody was obtained from Life Technologies
Summary
DNA double strand breaks (DSBs) are highly deleterious events that may lead to chromosomal abnormalities, cell death and cancer. An initial and essential step in HR is the 5 to 3 resection of the dsDNA at the DSB end, which leaves 3 single-stranded DNA (ssDNA) tails. Both in vivo and in vitro evidence suggests that resection is initiated by the Mre11-Rad50-Xrs complex (MRX) together with Sae, the budding yeast homolog of CtIP [5,6,7,8]. If only one end of the DSB is capable of pairing with homologous sequences, repair proceeds by a recombination-dependent process termed break-induced replication (BIR) [18,19]. Repair can occur in a Rad51independent fashion by single-strand annealing (SSA) when there are homologous sequences flanking a DSB [20]
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