Non-B DNA-forming Sequences and WRN Deficiency Independently Increase the Frequency of Base Substitution in Human Cells

Abstract

Although alternative DNA secondary structures (non-B DNA) can induce genomic rearrangements, their associated mutational spectra remain largely unknown. The helicase activity of WRN, which is absent in the human progeroid Werner syndrome, is thought to counteract this genomic instability. We determined non-B DNA-induced mutation frequencies and spectra in human U2OS osteosarcoma cells and assessed the role of WRN in isogenic knockdown (WRN-KD) cells using a supF gene mutation reporter system flanked by triplex-or Z-DNA-forming sequences. Although both non-B DNA and WRN-KD served to increase the mutation frequency, the increase afforded by WRN-KD was independent of DNA structure despite the fact that purified WRN helicase was found to resolve these structures in vitro. In U2OS cells, ϳ70% of mutations comprised single-base substitutions, mostly at G⅐C basepairs, with the remaining ϳ30% being microdeletions. The number of mutations at G⅐C base-pairs in the context of NGNN/ NNCN sequences correlated well with predicted free energies of base stacking and ionization potentials, suggesting a possible origin via oxidation reactions involving electron loss and subsequent electron transfer (hole migration) between neighboring bases. A set of ϳ40,000 somatic mutations at G⅐C base pairs identified in a lung cancer genome exhibited similar correlations, implying that hole migration may also be involved. We conclude that alternative DNA conformations, WRN deficiency and lung tumorigenesis may all serve to increase the mutation rate by promoting, through diverse pathways, oxidation reactions that perturb the electron orbitals of neighboring bases. It follows that such "hole migration" is likely to play a much more widespread role in mutagenesis than previously anticipated.The application of high resolution array comparative genomic hybridization techniques has revealed the frequent occurrence of DNA sequence motifs capable of forming alternative (non-B) DNA conformations (e.g. triplex, quadruplex, Z-DNA, cruciforms, slipped structures) at the breakpoint junctions of chromosomal alterations (gross deletions and duplications) associated with human genetic disease, including cystic fibrosis, mental retardation, and multiple congenital anomalies (1). These observations have served to extend the generality of previous work that aimed to elucidate the molecular mechanisms underlying recurrent translocations (2-6) and the genetic instability observed in many model systems (7-16), both of which were suggestive of a direct mutagenic role for non-B DNA. In the same vein, analyses of DNA sequence motifs flanking human gross deletion breakpoints (9), genomic inversions that distinguish the human from the chimpanzee genome (17), and DNA sequence tracts involved in pathological gene conversion events (18) have provided evidence for a wide ranging role for DNA secondary structure in promoting gross genomic rearrangements. Despite these recent advances, few studies (8, 11) have attempted to address systematically the extent of the influence ...

Exploring the somatic NF1 mutational spectrum associated with NF1 cutaneous neurofibromas

“…Several repetitive elements were found in the vicinity of these juxtaposed mutations: for example, an inverted repeat and a symmetric element were found in the vicinity of the c.1177-8 mutations; a direct repeat and run of identical nucleotides span the site of the c.5894 mutations, whereas c.6409 occurs in the vicinity of symmetric elements. These repeat sequences may serve to facilitate the formation of multiple non-B DNA structures, 48 thereby accounting for the hypermutability of these sites (shaded tumour IDs in Table 1). Previous mutation studies have also purportedly found evidence for potential clustering of mutations identified in separate neurofibromas from the same patient, 48,49 but such conclusions have never before received formal statistical support.…”

“…These repeat sequences may serve to facilitate the formation of multiple non-B DNA structures, 48 thereby accounting for the hypermutability of these sites (shaded tumour IDs in Table 1). Previous mutation studies have also purportedly found evidence for potential clustering of mutations identified in separate neurofibromas from the same patient, 48,49 but such conclusions have never before received formal statistical support. Although it is possible that the germline NF1 mutation might influence the location of subsequent somatic NF1 mutations, our current study on a relatively small number of paired germline somatic mutations have provided no evidence to support this postulate.…”

The Werner Syndrome Exonuclease Facilitates DNA Degradation and High Fidelity DNA Polymerization by Human DNA Polymerase δ

“…We show that, unlike wild-type cells, cells that lack WRN exhibit a 5-fold elevated mutation frequency during extension of synthetic DNA substrates in vivo. Using a completely different reporter assay, Bacolla et al (43) also recently noted that WRN deficiency increases the frequency of base substitution errors 2-fold in human cells. This spontaneous increase in mutation frequency may be low, but it is observed in unstressed cells that are proficient for Pol ␦ and ⑀ proofreading.…”

The Gratuitous Repair on Undamaged DNA Misfold

“…It has been reported that, without a functional RecQ helicase, DNA replication does not advance normally. In humans, lacking of WRN or BLM protein accumulates aberrant replication intermediates (Harrigan et al, 2003;Cheok et al, 2005), this may allow for certain non-B DNA structure forming (Mohaghegh et al, 2001;Bacolla et al, 2011). Therefore, it is not surprising to see that more and more reports are going to be published www.intechopen.com…”