The Replication Checkpoint Prevents Two Types of Fork Collapse without Regulating Replisome Stability

Abstract

Summary The ATR replication checkpoint ensures that stalled forks remain stable when replisome movement is impeded. Using an improved iPOND protocol combined with SILAC mass spectrometry, we characterized human replisome dynamics in response to fork stalling. Our data provide a quantitative picture of the replisome and replication stress response proteomes in 32 experimental conditions. Importantly, rather than stabilize the replisome, the checkpoint prevents two distinct types of fork collapse. Unsupervised hierarchical clustering of protein abundance on nascent DNA is sufficient to identify protein complexes and place newly identified replisome-associated proteins into functional pathways. As an example, we demonstrate that ZNF644 complexes with the G9a/GLP methyltransferase at replication forks and is needed to prevent replication-associated DNA damage. Our data reveal how the replication checkpoint preserves genome integrity, provide insights into the mechanism of action of ATR inhibitors, and will be a useful resource for replication, DNA repair, and chromatin investigators.

TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

“…1 H), further suggesting that TRA IP associates with the replication machinery. Despite the fact that NCC analysis revealed a strong net loss of PCNA from chromatin upon hydroxyurea (HU) treatment, which has also been observed in isolation of proteins on nascent DNA (iPOND) experiments (Sirbu et al, 2011;Dungrawala et al, 2015), the levels of TRA IP essentially mirrored this behavior ( Fig. 1 H), further suggesting a close link between TRA IP and replisome-associated PCNA.…”

“…One attractive, though highly speculative, possibility is therefore that TRA IP-mediated ubiquitylation of PCNA could help to promote its extraction or unloading from stalled forks as part of responses that remodel the DNA replication machinery to overcome replication blocks. To this end, experiments using isolation of proteins on nascent DNA (iPOND; Sirbu et al, 2011;Dungrawala et al, 2015), enriching and sequencing of protein-associated nascent strand DNA (eSPAN; Yu et al, 2014), and our NCC experiments have demonstrated rapid loss of overall PCNA levels from the replication machinery upon fork stalling induced by short treatment with HU in both yeast and human cells. It remains to be determined whether this is simply a consequence of reduced loading of PCNA on the lagging strand without a concomitant decrease in the rate of unloading, or whether the loss of PCNA can also be mediated by its active displacement from stalled forks.…”

DNA damage-induced ATM- and Rad-3-related (ATR) kinase activation in non-replicating cells is regulated by the XPB subunit of transcription factor IIH (TFIIH)

“…Consistent with this hypothesis, TFIIH subunits were found by mass spectrometry to accumulate on nascent DNA at stalled replication forks (69). Preliminary studies using a sensitive assay for detecting the small excised oligonucleotide products of nucleotide excision repair (70-73) have thus far indicated that TFIIH function in this context is likely independent of nucleotide excision repair (Kemp, unpublished data).…”

The expanding role of the Ehmt2/G9a complex in neurodevelopment

“…Alternatively, this S-phase delay may be in part due to a recently described role of the Ehmt2 complex at the replication fork. Znf644, Ehmt1 and Ehmt2 are present at the replication fork, and their removal resulted in phenotypes consistent with replication stress, 83 perhaps owing to impaired Pcna loading. While the role of the Ehmt1/2 complex at the replication fork is still not defined, it was suggested that it may be necessary to regulate non-histone protein function at the fork, or for transmission of H3K9me2 marks to newly synthesized chromatin.…”