Mutagenic Capacity of Endogenous G4 DNA Underlies Genome Instability in FANCJ-Defective C. elegans

Abstract

To safeguard genetic integrity, cells have evolved an accurate but not failsafe mechanism of DNA replication. Not all DNA sequences tolerate DNA replication equally well [1]. Also, genomic regions that impose structural barriers to the DNA replication fork are a potential source of genetic instability [2, 3]. Here, we demonstrate that G4 DNA-a sequence motif that folds into quadruplex structures in vitro [4, 5]-is highly mutagenic in vivo and is removed from genomes that lack dog-1, the C. elegans ortholog of mammalian FANCJ [6, 7], which is mutated in Fanconi anemia patients [8-11]. We show that sequences that match the G4 DNA signature G3-5N1-3G3-5N1-3G3-5N1-3G3-5 are deleted in germ and somatic tissues of dog-1 animals. Unbiased aCGH analyses of dog-1 genomes that were allowed to accumulate mutations in >100 replication cycles indicate that deletions are found exclusively at G4 DNA; deletion frequencies can reach 4% per site per animal generation. We found that deletion sizes fall short of Okazaki fragment lengths [12], and no significant microhomology was observed at deletion junctions. The existence of 376,000 potentially mutagenic G4 DNA sites in the human genome could have major implications for the etiology of hereditary FancJ and nonhereditary cancers.

A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites

“…With its 22-kb open reading frame (ORF) and a clearly recognizable mutant phenotype, the unc-22 locus provides an endogenous mutational sink that is largely non-discriminatory to size: all alterations that change the reading frame (whether one bp or tens of kb) will render worms insensitive to the muscle hypercontracting effect of the cholinergic agonist levamisole 21 . Earlier work revealed that sequences that match the G4 DNA consensus G 3-5 N 1-3 G 3-5 N 1-3 G 3-5 N 1-3 G 3-5 induce deletions in dog-1-deficient animals; equienergetic G-rich DNA sequences that do not match this motif (for example, G3C DNA or CG repeats) are perfectly stable 17 . However, because of the relatively high, size-related, background level of spontaneous mutagenesis in unc-22 (B10 À 6 ), we chose to target only those G4 sequences that we previously found to have high mutagenic potential (Fig.…”

“…1a). As of the low mutagenic capacity of minimal G4 motifs 17 , we obtained qua739 deletions at its original genomic location using a PCR-based approach (see Methods section). Indeed, deletions triggered by this motif have a very sharply positioned 3 0 junction, with none mapping within the motif (Fig.…”

“…Later work demonstrated that only G-rich DNA sequences that match the signature G4 motif G 3-5 N 1-3 G 3-5 N 1-3 G 3-5 N 1-3 G 3-5 define sites of genomic instability 17 ; G-rich DNA unable to adopt a G-quadruplex structure remained perfectly stable in DOG-1-deficient worms. An evolutionary conserved role for DOG-1/ FANCJ in preventing G4 DNA-induced genome alteration was suggested by the mapping of large genomic deletions accumulating in a FANCJ-deficient human patient cell line to G4 DNA-containing regions 18 .…”

“…In line with a proposed role for this helicase in resolving G-quadruplexes in vivo to help the fork replicate the affected strand 16 , the purified human FANCJ protein was shown to behave as a structure-specific DNA helicase that can unwind G4 DNA with 5 0 to 3 0 polarity 19 . While the molecular nature of genomic alterations in C. elegans demonstrated a strikingly atypical mutation profile 16,17 , little is known about the molecular mechanisms that underlie G4 DNA-induced genome rearrangements. Here we identify an alternative DNA doublestrand break (DSB) repair pathway, which requires Polymerase Theta (y), and which is the pathway of choice to cope with these structural replication fork barriers.…”

“…In addition, Drosophila Pol y was recently implicated in the repair of DNA DSBs induced by DNA transposition 28 . To test a possible involvement of the C. elegans homologue of Pol y, POLQ-1, we first used a PCR-based assay that detects G4 DNA-induced deletions in isolated DNA by preferential amplification of smaller than wild-type bands of sequences containing G4 motifs 17 . We found that, while 14% of dog-1 animals sustained at least one small-sized deletion at the G4 motif qua830, none were detected in animals that in addition were also deficient for polq-1 (Fig.…”