Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites

Shyam Twayana,Albino Bacolla,Angelica Barreto-Galvez,Ruth B De-Paula,William C Drosopoulos,Settapong T Kosiyatrakul,Eric E Bouhassira,John A Tainer,Advaitha Madireddy,Carl L Schildkraut

doi:10.1073/pnas.2106477118

Abstract

Significance Common fragile sites (CFSs) are normal loci that are genetically unstable under normal and oncogenic replication stress. Pol eta has been proposed to play a key role in CFS replication. Here, we show that in the absence of Pol eta, replication at five specific CFS loci is perturbed, with fork pausing observed at several sites. Sequence analysis showed that certain pause sites are associated with the presence of non-B DNA motifs, while others are not. Importantly, pause sites are located within regions of increased genetic variation in healthy human populations that could be attributed to Pol eta activity. Our data unveil a role for Pol eta in overcoming replication stress, reducing DNA breakage, and promoting genetic variation at CFSs.

Highlights

Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress
The replicative DNA Pol delta pauses at CFS-associated repeat sequences in vitro [19, 20], and translesion synthesis (TLS) Pols like Pol eta are postulated to exchange with paused Pol delta to complete DNA synthesis at CFS-associated repetitive sequences [21]
Pol eta is recruited to CFSs in vivo and can efficiently replicate through CFS-associated repetitive sequences in vitro [13, 21]

Summary

Introduction

Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. In Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. Pol eta is recruited to CFS loci upon replication stress and could replicate CFS-associated non-B DNA sequences in vitro [13]. Using our powerful locus-specific single-molecule analysis of replicated DNA (SMARD) approach, we previously identified important factors affecting replication programs at genomic regions containing repetitive sequences within rare fragile sites [22, 23], telomeres [24, 25], CFSs [9], and episomal Kaposi sarcoma-associated herpesvirus [26]. Significance common fragile sites j polymerase eta j replication fork pause j non-B DNA j SNP

Methods

Results

Conclusion