Clusters of Multiple Mutations: Incidence and Molecular Mechanisms

Abstract

It has been long understood that mutation distribution is not completely random across genomic space and in time. Indeed, recent surprising discoveries identified multiple simultaneous mutations occurring in tiny regions within chromosomes while the rest of the genome remains relatively mutation-free. Mechanistic elucidation of these phenomena, called mutation showers, mutation clusters, or kataegis, in parallel with findings of abundant clustered mutagenesis in cancer genomes, is ongoing. So far, the combination of factors most important for clustered mutagenesis is the induction of DNA lesions within unusually long and persistent single-strand DNA intermediates. In addition to being a fascinating phenomenon, clustered mutagenesis also became an indispensable tool for identifying a previously unrecognized major source of mutation in cancer, APOBEC cytidine deaminases. Future research on clustered mutagenesis may shed light onto important mechanistic details of genome maintenance, with potentially profound implications for human health.

Keywords: cancer; evolution; genome instability; kataegis; mutagenesis; mutation showers.

Figure 1. Complex mutations can be generated by a single error or obstruction in DNA copying

(a) Misincorporation by error-prone TLS polymerase. Initiating event: A TLS polymerase inserts a mismatched C (in blue) opposite a damaged T (in orange, with asterisk). Step (i): The TLS polymerase synthesizes several more bases and misincorporates another base (A opposite undamaged C), generating a complex mutation event. (b) Slippage by error-prone TLS polymerase. Initiating event: A TLS polymerase inserts a mismatched A opposite a damaged G. Step (i): The polymerase synthesizes further, encountering a T homonucleotide run. Slippage within the run produces a single-nucleotide bulge. (ii) Synthesis continues beyond homonucleotide run, leaving the bulge uncorrected. Adapted from Figure 2 in (49). (c) A replicase is blocked from further synthesis at a hairpin formed by short inverted repeat. Step (i): A TLS polymerase bypasses the blocking hairpin by switching template and synthesizing a short stretch (in orange). Step (ii): The newly extended nascent strand then realigns to original template, despite mismatches to the resolved hairpin (bulging nucleotides). A replicase continues synthesis beyond the mismatches, yielding a complex mutation event. Adapted from Figure 6 in (85).

Figure 2

Mutation clusters are abundant across multiple cancer types. Clusters are categorized as: perfectly A- or T-coordinated, C- or G-coordinated, or non-coordinated. Note that nearly half of C- or G-coordinated clusters are found <20 kb from a breakpoint junction between two rearranged chromosomes. Adapted from (107; 108).

Figure 3. Signature of APOBEC mutagenesis in clusters of a breast cancer genome

(a) 18 perfectly C- or G-coordinated clusters with >6 mutations from breast cancer sample PD4103a (83) are lined up, with the first mutation assigned as relative base position 1. Mutations are numbered consecutively, from the first mutation in the short arm of chromosome 1 to the last mutation in the long arm of chromosome X. Numbers listed along vertical axis denote first and last mutations that comprise each cluster. (b) All base substitutions from PD4103a are shown in a “rainfall” plot, where distance between adjacent mutations is plotted on a log scale vs. mutation number (mutation numbers defined as in (a)). Some regions of “kataegis” are indicated by arrowheads. There are many more clusters within the kataegic regions than shown in (a), including shorter and/or not perfectly C- or G-coordinated examples. (c) C- or G-Coordinated clusters consist predominantly of tCw -> tGw (42.1%) or tCw -> tTw (44.6%) mutations. tCw -> tAw represent only 13.3%.

Figure 4. Mutation clusters resulting from lesions that had opportunity for templated repair

(a) Lack of lesion repair in a region of dsDNA. In presence of mutagen, base damage occurs in both strands of dsDNA. Step (i): Replication of unrepaired template strands results in TLS-mediated incorporation of mismatches opposite the lesions. Step (ii): Repair after replication fixes mutation clusters. If a mutagen is base-specific, there will be reciprocal strand-coordination in the two daughter duplexes. Step (iii): Repair occurs before replication. Step (iv): Undamaged templates are replicated. Step (v): Daughter duplexes contain no mutations. (b) R-loops. A nascent mRNA (in green) remains annealed to its DNA template, generating an R-loop. The displaced ssDNA is damaged. Step (i): A persistent R-loop would block excision repair and replication would proceed on a damaged template top strand. Step (ii): TLS and excision repair fix mutation cluster in a daughter duplex. Step (iii): If the mRNA is removed, the DNA duplex re-anneals, enabling excision repair of damage. Step (iv) Replication of the repaired template is mutation-free. (c) Lesions in ssDNA of the lagging strand. Perturbation of lagging strand synthesis exposes portion of template ssDNA. Green squares denote RNA primers. Exposed ssDNA sustains base damage. Step (i): Error-prone TLS. Step (ii) Subsequent excision repair fixes mutation cluster in a daughter duplex. Step (iii): Fork regression repositions lesions into dsDNA section, where excision repair can occur. Step (iv): Damage is repaired. Step (v): Replication occurs on undamaged template, resulting in mutation-free daughter duplexes.

Figure 5. Mutation clusters resulting from lesions in ssDNA without an opportunity for templated repair

(a) Clusters associated with 5′→3′ resection at uncapped telomeres. Step (i): The protective cap of a telomere dissociates. The uncapped telomere is recognized as a DSB and is resected to generate long 3′ ssDNA overhang. Base damage (orange stars) occurs within the exposed ssDNA, e.g., APOBEC generating uracils. Step (ii): If uracils are not excised, a replicase simply inserts adenines. Step (iii): Base excision repair excises uracils and uses the adenines to template repair, resulting in cluster of C to T transitions. Alternatively, DNA with U:A pairs can be copied without base excision repair, still producing a product with C to T transitions. Step (iv): If uracils created by C-deamination in ssDNA are excised by uracil DNA glycosylase, abasic (AP) sites are generated. Step (v): AP sites are bypassed in an error-prone manner by TLS polymerases, usually inserting either A or C across AP-sites. Step (vi): Excision repair fixes mutation cluster containing a mix of C to T transitions and C to G transversions. (b) Clusters associated with 5′→3′ resection around a DSB. Step (i): A two-sided DSB is resected to generate 3′ ssDNA overhangs. Base damage at e.g. cytosines (base specificity shown by orange color) occurs within overhangs. Step (ii): Repair synthesis, using TLS polymerase(s) inserts bases opposite the lesions in an error-prone way. Step (iii): Excision repair fixes mutation cluster spanning the DSB region. Note the switch in base specificity of strand-coordination within these clusters. (c) Clusters associated with ssDNA generated by break-induced replication (BIR). Step (i): The 3′ ssDNA overhang from a resected one-sided DSB invades a homologous sequence in a donor chromosome. Step (ii): Leading strand synthesis (solid arrow) proceeds, while lagging strand synthesis (dashed arrow) occurs later, exposing long stretches of ssDNA, which sustain base damage. Step (iii): Completion of lagging strand synthesis and excision repair fix mutation cluster in repaired chromosome. Note that BIR-associated clusters do not show a switch in base specificity of strand-coordination (compare (c) with (b))

Figure 6

A mutation cluster caused by chronic damage to DNA of proliferating yeast cells (108). Whole genome sequencing of yeast exposed chronically to MMS revealed a large strand-coordinated cluster of 26 mutations, which extended for ~200 kb including the CAN1-URA3 double reporter gene region (red bar), illustrating the hypermutable nature of exposed ssDNA. Strikingly, the rest of genome harbored only 19 mutations.