Nearly 30 hereditary disorders in humans result from an increase in the number of copies of simple repeats in genomic DNA. These DNA repeats seem to be predisposed to such expansion because they have unusual structural features, which disrupt the cellular replication, repair and recombination machineries. The presence of expanded DNA repeats alters gene expression in human cells, leading to disease. Surprisingly, many of these debilitating diseases are caused by repeat expansions in the non-coding regions of their resident genes. It is becoming clear that the peculiar structures of repeat-containing transcripts are at the heart of the pathogenesis of these diseases.

A DNA triplex is formed when pyrimidine or purine bases occupy the major groove of the DNA double Helix forming Hoogsteen pairs with purines of the Watson-Crick basepairs. Intermolecular triplexes are formed between triplex forming oligonucleotides (TFO) and target sequences on duplex DNA. Intramolecular triplexes are the major elements of H-DNAs, unusual DNA structures, which are formed in homopurine-homopyrimidine regions of supercoiled DNAs. TFOs are promising gene-drugs, which can be used in an anti-gene strategy, that attempt to modulate gene activity in vivo. Numerous chemical modifications of TFO are known. In peptide nucleic acid (PNA), the sugar-phosphate backbone is replaced with a protein-like backbone. PNAs form P-loops while interacting with duplex DNA forming triplex with one of DNA strands leaving the other strand displaced. Very unusual recombination or parallel triplexes, or R-DNA, have been assumed to form under RecA protein in the course of homologous recombination.

SUMMARY Accurate and complete replication of the genome in every cell division is a prerequisite of genomic stability. Thus, both prokaryotic and eukaryotic replication forks are extremely precise and robust molecular machines that have evolved to be up to the task. However, it has recently become clear that the replication fork is more of a hurdler than a runner: it must overcome various obstacles present on its way. Such obstacles can be called natural impediments to DNA replication, as opposed to external and genetic factors. Natural impediments to DNA replication are particular DNA binding proteins, unusual secondary structures in DNA, and transcription complexes that occasionally (in eukaryotes) or constantly (in prokaryotes) operate on replicating templates. This review describes the mechanisms and consequences of replication stalling at various natural impediments, with an emphasis on the role of replication stalling in genomic instability.

Eukaryotic DNA replication is highly stratified, with different genomic regions shown to replicate at characteristic times during S phase. Here we observe that mutation rate, as reflected in recent evolutionary divergence and human nucleotide diversity, is markedly increased in later-replicating regions of the human genome. All classes of substitutions are affected, suggesting a generalized mechanism involving replication time-dependent DNA damage. This correlation between mutation rate and regionally stratified replication timing may have substantial evolutionary implications.Evolutionary divergence and inferred mutation rates are known to vary across the human genome [1][2][3] , and it has long been speculated that this is a consequence of covariance with an epigenetic feature1 , 2. In human cells, the time of DNA replication exhibits marked regional variability during an S-phase lasting approximately 10-hours4 ,5 . To parallel the conventional division of S-phase into four sequential temporal states (S1-S4), we used a hidden Markov model6 to perform unbiased four-state partitioning of continuous, highresolution replication timing measurements across 1% of the human genome7. We then determined human-chimpanzee nucleotide divergence rates and the density of SNPs8 at putatively neutrally evolving sites within each temporal state, excluding any bases within annotated exons, repetitive elements, CpG islands, 2kb-regions upstream and downstream of genes, intronic splice sites, and conserved non-coding sequences9 (Supplementary Table S1).We observed a striking trend relating the rate of evolutionary divergence and the density of human SNPs to the progress of DNA replication (Fig. 1). Human-chimpanzee substitutions and human SNP density increase 22% and 53%, respectively, during the temporal course of replication, both of which are highly statistically significant (p < 8.43 × 10 −26 , CochranArmitage; Fig. 1a-c, g-i). To rule out potential confounding by the overall low genomewide rate of human-chimpanzee divergence, we also analyzed human-macaque divergence, with similar results (p < 2.7 × 10 −54 ; Fig. 1d-f). We confirmed the absence of bias due to a sampling or stratification effect across different genomic regions by testing (CochranMantel-Haenszel) for three-way interactions, treating region assignment as controlling variable (p < 7.2 × 10 −12 , p < 0.00026 for human-chimpanzee divergence and human SNPs, NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript respectively). Additionally, we repeated all analyses with an independent set of randomly ascertained SNPs 10 , with nearly identical effect (p < 9.69 × 10 −22 ).Next we examined whether the observed correlation between mutation rate and replication time could be explained by variation in another genomic feature for which replication timing might be acting as a surrogate. Regional variation in G+C content 2,3 and, independently, recombination rate2 , 3 have been invoked as potential causes of human mutation rate variation. We the...

(CGG)n.(CCG)n and (CTG)n.(CAG)n repeats of varying length were cloned into a bacterial plasmid, and the progression of the replication fork through these repeats was followed using electrophoretic analysis of replication intermediates. We observed stalling of the replication fork within repeated DNAs and found that this effect depends on repeat length, repeat orientation relative to the replication origin and the status of protein synthesis in a cell. Interruptions within repeated DNAs, similar to those observed in human genes, abolished the replication blockage. Our results suggest that the formation of unusual DNA structures by trinucleotide repeats in the lagging-strand template may account for the observed replication blockage and have relevance to repeat expansion in humans.