Duplex DNA from Sites of Helicase-Polymerase Uncoupling Links Non-B DNA Structure Formation to Replicative Stress

Abstract

Background: Replication impediments can produce helicase-polymerase uncoupling allowing lagging strand synthesis to continue for as much as 6 kb from the site of the impediment. Materials and Methods: We developed a cloning procedure designed to recover fragments from lagging strand near the helicase halt site. Results: A total of 62% of clones from a p53-deficient tumor cell line (PC3) and 33% of the clones from a primary cell line (HPS-19I) were within 5 kb of a G-quadruplex forming sequence. Analyses of a RACK7 gene sequence, that was cloned multiple times from the PC3 line, revealed multiple deletions in region about 1 kb from the cloned region that was present in a non-B conformation. Sequences from the region formed G-quadruplex and i-motif structures under physiological conditions. Conclusion: Defects in components of non-B structure suppression systems (e.g. p53 helicase targeting) promote replication-linked damage selectively targeted to sequences prone to G-quadruplex and i-motif formation. Non-B DNA structure formation must be suppressed during replication in order for replication to proceed properly. Many such structures occur at DNA sequences that exhibit GC-skew where cytidine residues are largely confined to one strand and guanidine residues are largely confined to the other. In addition to R-Loops (1), these sequences are capable of forming G-quadruplexes (2, 3) triple helices (4) and i-motifs (5). At physiological pH, certain sequences are capable of forming several different structures (6, 7), however, the most commonly seen structures in these regions are the R-Loop, G-quadruplex and, less frequently, the triple helix (7) and the i-motif (8). DNA damage associated with R-loop formation is generally associated with transcription blocks (9, 10), but can be associated with replication. There is also considerable evidence that G-quadruplex formation can cause both epigenetic (11, 12) and genetic damage associated with DNA replication when G-quadruplex suppression systems are compromised (13-15). Here it is important to point out that G4-seq (60) results confirm the earlier report (16) that the frequency of G-quadruplex forming sequences increases with biological complexity suggesting that these sequences have been under positive selective pressure during evolution of higher organisms. To facilitate this increased frequency, the systems that promote replication through G-quadruplex structures or unwind these structures during replication and transcription of DNA appear to have evolved increased sophistication and redundancy in higher organisms. For example the Y polymerases REV1, Pol ĸ and Pol η are known to process G-quadruplex motifs when replication is slowed by them (17). Further, several helicases including Pif1 (15), and the superfamily 2 helicases WRN (18), BLM (19) and FANCJ (20) have all been shown to unwind G-quadruplex DNA structures in vitro. Even so, WRN (21), BLM (22), Pif1 (23), and FNACJ (24) are also capable of unwinding RNA:DNA hybrids in vitro.

Replication‐induced DNA secondary structures drive fork uncoupling and breakage

“…Similar to our previous work with repetitive sequences (Casas‐Delucchi et al , 2022), we observe uncoupling between helicase unwinding and DNA synthesis in response to quadruplexes. This is consistent with previous work which demonstrates an enrichment of G4‐forming sequences within 6 kb of uncoupled forks in tumour cells (Amparo et al , 2020). This response is akin to the response to leading strand DNA damage (Taylor & Yeeles, 2018) and leads to the exposure of ssDNA.…”

Replication-induced DNA secondary structures drive fork uncoupling and breakage

“…Similar to previous work investigating the effects of repetitive sequences on eukaryotic replication 45 , we observe uncoupling between helicase unwinding and DNA synthesis in response to both i-motifs and G4s. This is consistent with previous work which demonstrates an enrichment of G4-forming sequences within 6kb of uncoupled forks in tumour cells 75 . This response is akin to the response to leading strand DNA damage 54 and leads to the exposure of ssDNA.…”

Remodeling the conformational dynamics of I-motif DNA by helicases in ATP-independent mode at acidic environment

“…In addition, Amparo et al. discovered that the formation of non-B structures including G4 and i-motif could induce the helicase-polymerase uncoupling, causing the replicative stress ( Amparo et al., 2020 ). In cells, helicases assist DNA polymerase to replicate the structured DNAs, and a number of helicases including Pif1, BLM, DHX36, and WRN have been shown to resolve G4s both in vivo and in vitro .…”

“…More importantly, how the folding conformation of i-motif DNA is regulated by these proteins remains elusive. It is worth noting that a recent study linked the formation of G4s and i-motifs in the helicase-polymerase uncoupling sites with the replicative stress and suggested the requirement of DNA repair ( Amparo et al., 2020 ). We know that helicases participate in almost every aspect of DNA metabolism; particularly, some helicases are responsible for resolving the complicated DNA structures like G4s and Holliday junctions ( Sharma, 2011 ).…”