The Saccharomyces cerevisiae Rad6 Postreplication Repair and Siz1/Srs2 Homologous Recombination-Inhibiting Pathways Process DNA Damage That Arises in asf1 Mutants

Abstract

The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.DNA replication must be highly coordinated with chromatin assembly and cell division for correct propagation of genetic information and cell survival. Errors arising during DNA replication are corrected through the functions of numerous pathways including checkpoints and a diversity of DNA repair mechanisms (32,33,35). However, in the absence of these critical cellular responses, replication errors can lead to the accumulation of mutations and gross chromosomal rearrangements (GCRs) as well as chromosome loss, a condition generally termed genomic instability (33). Genome instability is a hallmark of many cancers as well as other human diseases (24). There are many mechanisms by which GCRs can arise, and over the last few years numerous genes and pathways have been implicated in playing a role in the suppression of GCRs in Saccharomyces cerevisiae and in some cases in the etiology of cancer (27, 28, 33, 39-47, 51, 53, 56, 58, 60), including S. cerevisiae ASF1, which encodes the main subunit of the replication coupling assembly factor (37, 62).Asf1 is involved in the deposition of histones H3 and H4 onto newly synthesized DNA during DNA replication and repair (62), and correspondingly, asf1 mutants are sensitive to chronic treatment with DNA-damaging agents (2, 30, 62). However, asf1 mutants do not appear to be repair defective and can recover from acute treatment with at least some DNAdamaging agents (2,8,30,31,54), properties similar to those described for rad9 mutants (68). In the absence of Asf1, both the DNA damage and replication checkpoints become activated during normal cell growth, and in the absence of checkpoint execution, there is a further incr...

Distinct SUMO Ligases Cooperate with Esc2 and Slx5 to Suppress Duplication-Mediated Genome Rearrangements

“…In contrast, Siz1, a SUMO E3 ligase [11], does not have a significant role in suppressing duplication-mediated GCRs [12]. However, deletion of SIZ1 suppresses the GCR defect of asf1 mutant mediated by single-copy sequences to essentially wild-type levels [13]. Taken together, these observations raise the possibility that protein sumoylation may have a role in regulating and in some cases suppressing GCRs.…”

Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes

“…Cell lysis, immunoprecipitation, and Western blotting IP and Western blotting was performed as previously described (Sacher et al 2005;Kats et al 2009;Chymkowitch et al 2012). All buffers contained N-Ethylmaleimide to prevent desumoylation.…”

TORC1-dependent sumoylation of Rpc82 promotes RNA polymerase III assembly and activity

“…IP and Western blotting was performed as previously described (26,29,30). All buffers contained 10 mM of fresh N-ethylmaleimide (Sigma) to prevent desumoylation by SUMO proteases.…”