Genome-wide Identification of Structure-Forming Repeats as Principal Sites of Fork Collapse upon ATR Inhibition

Abstract

Summary DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods (RPA-ChIP and BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these categories of repeats differed both in structure formation and in their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.

The sequence of the extruded non-template strand determines the architecture of R-loops

“…We speculate that structured R-loops could be more toxic for the cell and become replication impediments, as proposed recently (21). Consistent with this hypothesis, the formation of R-loops at Airn was shown to induce replication stress (4) and structure forming non-B DNA sequences are prominent barriers to replication fork progression (21,27). Structured R-loops could impede directly the passage of replication forks.…”

The role of fork stalling and DNA structures in causing chromosome fragility

“…The intergenic breaks at poly (dA:T) sites were present both when cells were exposed to a low dose of HU or no exogenous replication stress, emphasizing the role of sequence in their fragility. In contrast, a study that mapped DSBs by a different technique (BrITL) in conditions of ATR inhibition did not identify mononucleotide A:T runs, but rather break sites were enriched at structure‐forming repeats in both mouse and human cells (covered below) …”

“…This study reinforces the link between DNA structures and fragility and indicates that the exact type of structure and base composition can differ among organisms. Interestingly, poly (dA:T) sequences were not enriched in this study as they were in Ref. .…”

“…In contrast, a study that mapped DSBs by a different technique (BrITL) in conditions of ATR inhibition did not identify mononucleotide A:T runs, but rather break sites were enriched at structure-forming repeats in both mouse and human cells (covered below). 48 Another interesting sequence is the ATTCT repeat at the ATXN10 locus which can expand to cause spinocerebellar ataxia type 10.…”

“…117,118 A recent genome-wide analysis of break sites in conditions of ATR inhibition alone or in combination with low-dose aphidicolin was accomplished using two different methods: RPA ChIP and a new highresolution break detection method, Breaks Identified by TdT Labeling (BrITL). 48 In mouse embryonic fibroblasts, there was a strong enrichment for microsatellite repeats CAGAGG and CACAG (and derivatives), which were shown to form a structure and pause replication, as well as IRs and quasi-palindromes. In human breast cancer cells, structure-forming repeats were also highly enriched at break sites, but interestingly the repeat sequences were different from those identified in mouse cells.…”