Hypermutation in single-stranded DNA

Abstract

Regions of genomic DNA can become single-stranded in the course of normal replication and transcription as well as during DNA repair. Abnormal repair and replication intermediates can contain large stretches of persistent single-stranded DNA, which is extremely vulnerable to DNA damaging agents and hypermutation. Since such single-stranded DNA spans only a fraction of the genome at a given instance, hypermutation in these regions leads to tightly-spaced mutation clusters. This phenomenon of hypermutation in single-stranded DNA has been documented in several experimental models as well as in cancer genomes. Recently, hypermutated single-stranded RNA viral genomes also have been documented. Moreover, indications of hypermutation in single-stranded DNA may also be found in the human germline. This review will summarize key current knowledge and the recent developments in understanding the diverse mechanisms and sources of ssDNA hypermutation.

Keywords: Cancer; Hypermutation; Mutation clusters; Single-stranded DNA.

Fig. 1

Hypermutation in ssDNA formed during DSB repair. (The color code and symbols explained in the legend to panel A are used throughout in all panels of Figs. 1–3). The figures are adapted from [7]. A. 5′ to 3′ DNA resection at uncapped telomeres. (i) In the temperature sensitive cdc13-1 yeast strains, yeast telomeres are uncapped upon shift of the strains to 37 °C. 5′ to 3′ resection ensues and approximately 30 kb of the telomeric ends are rendered single stranded. The undamaged nucleotides are depicted as red (cytosines) or blue (guanine) circles with white background with the nucleotide shown as a black letter inside a circle. (ii) Exposure to a ssDNA-specific mutagen will lead to DNA damage in the un-resected single strand (yellow stars). In this figure, we depict DNA damage in cytosines in ssDNA as an example. (iii) Upon switch to permissive temperatures the resected strand is resynthesized. In the absence of excision of the damaged base by a glycosylase, DNA replication over the lesions will yield mutations. Mutated nucleotides are shown as filled circles. Red solid circles correspond to mutant nucleotides originated from damaged cytosines and blue solid circles to nucleotides that replaced damaged guanines. (The same color code and symbols are used in throughout all panels of Fig. 1, Fig. 2, Fig. 3). As an example, replication of unrepaired base damage in cytosines such as deamination will be expected to yield strand coordinated clusters (stretches of C→T changes). (iv) On the other hand, certain DNA glycosylases have been shown to function in ssDNA. As such, the removal of the damaged base (depicted as red circles with a yellow star) in ssDNA may lead to abasic sites in ssDNA (dotted red circles). (v, vi) Bypass of such abasic sites by translesion polymerases during resynthesis of the resected strand will lead to insertions of A or C opposite the abasic sites. In the example shown in the figure, bypass of abasic sites generated upon excision of damaged cytosines in ssDNA will yield C→T and C→G changes. B. Bidirectional resection at double strand breaks. (i) A two ended break is symmetrically resected on both sides of the break to generate 3′ overhangs. ssDNA-specific base damage in the overhangs and (ii, iii) bypass of the damaged base leads to single-switch clustered mutations. An example of ssDNA-specific damage leading to mutations in cytosines is shown here. DNA damage accumulated in the single stranded overhangs created during repair lead to clustered mutations in cytosines to the left of the break-point followed by clustered mutations in guanines to the right of the break-point. C. Break-induced replication. (i) A one-sided DNA double strand break is repaired via 5′ to 3′ resection of the broken end and (ii) a one-ended invasion into a homologous donor template. Uncoupling of the leading and lagging strands during break-induced replication yields long ssDNA intermediates that accumulate DNA damage. (iii) Synthesis of the second strand using the damaged template strand followed by excision repair of the lesions fixes the mutations in the newly synthesized DNA molecule. As an example, damage and mutations in cytosines is depicted here. Note that unlike for bi-directional resection, cytosines of the top strand are mutated on both sides of a DSB.

Fig. 2

Hypermutation in ssDNA formed during DNA replication and transcription. A. DNA damage in ssDNA formed on the lagging strand during DNA replication. A replication fork is shown. The arrows depict the direction of DNA synthesis. The green blocks are primers added by Polα. (i) In the presence of an ssDNA-specific DNA damaging agent, ssDNA gaps in the lagging strand accumulate DNA damage. As an example, DNA damage accumulated in cytosines is portrayed here. (ii, iii) Bypass of the lesions without excision repair fixes the mutations in the daughter DNA and leads to the formation of replication-strand biased mutation clusters. (iv, v) Alternatively, removal of the un-ligated Okazaki fragment by fork regression would place the damaged nucleotides in dsDNA allowing the damage to be repaired and replication would proceed over the undamaged template [122]. (vi) The daughter DNA molecules are not mutated. B. Perturbation of transcription may lead to stabilized R-loops leading to DNA damage accumulation in the single-stranded non-transcribed strand. The nascent RNA in the R-loop is shown in green. (i) As an example, ssDNA-specific mutagenesis in cytosines is shown in this figure. (ii) In the absence of repair, replication over the lesions leads to transcriptional-strand biased mutagenesis. Shown as C→T changes where the mutated cytosines are in the non-transcribed strand. (iii, iv) Resolution of the R-loops provides the undamaged template for excision repair and prevents mutagenesis.

Fig. 3

Strand-coordinated clusters from replication of unrepaired dsDNA. In the presence of a DNA damaging agent, lesions may be formed on both strands of dsDNA molecules. As an example, DNA damage in cytosines is depicted in this figure. (i) Lesions in cytosines are shown as yellow stars in red circles. (ii) Replication of the DNA molecules prior to repair leads to mismatches in the nascent DNA. (iii) Repair of lesions fixes the mutations in the daughter molecules. If the lesions were base specific (as shown here), the mutations will be reciprocally strand-coordinated in the daughter DNA molecules. (iv, v, vi) Alternatively, repair of lesions prior to DNA replication will prevent mutagenesis.