Error-prone bypass of DNA lesions during lagging-strand replication is a common source of germline and cancer mutations

Abstract

Studies in experimental systems have identified a multitude of mutational mechanisms including DNA replication infidelity and DNA damage followed by inefficient repair or replicative bypass. However, the relative contributions of these mechanisms to human germline mutation remain unknown. Here, we show that error-prone damage bypass on the lagging strand plays a major role in human mutagenesis. Transcription-coupled DNA repair removes lesions on the transcribed strand; lesions on the non-transcribed strand are preferentially converted into mutations. In human polymorphism we detect a striking similarity between mutation types predominant on the non-transcribed strand and on the strand lagging during replication. Moreover, damage-induced mutations in cancers accumulate asymmetrically with respect to the direction of replication, suggesting that DNA lesions are resolved asymmetrically. We experimentally demonstrate that replication delay greatly attenuates the mutagenic effect of ultraviolet irradiation, confirming that replication converts DNA damage into mutations. We estimate that at least 10% of human mutations arise due to DNA damage.

Figure 1.. R-asymmetry and T-asymmetry patterns in human polymorphism.

a, Relationship between R-asymmetry and T-asymmetry for 92 mutation types (NpCpG>T mutations excluded). b, Relationship between R-asymmetry and T-asymmetry shown separately for the six types of single-nucleotide mutations to highlight the effects of adjacent nucleotides.

Figure 2.. Damage-induced mutations preferentially reside on the lagging strand.

a, Number of tumor samples among melanomas, lung adeno carcinomas (LUAD), lung squamous carcinomas (LUSC), and liver cancers that have more damage-induced mutations on the leading than on the lagging strand (p-values shown for the goodness-of-fit chi-square test). b,c distribution of R-asymmetry (b) and T-asymmetry (c) values. Samples with T-asymmetry less than 1.2 were excluded from panel b.

Figure 3.. R-asymmetry in UV-irradiated cells.

R-asymmetry of repaired CPD damage (left) and CPD damage remaining in DNA (right) as a function of time since UV irradiation.

Figure 4.. Experimental design to test effect of replication delay on the rate of UV-induced mutations.

Clonal colonies of fibroblast cells shown in pink were treated with roscovitine for 3 hours in advance of the UV-irradiation. Colonies shown in blue were not treated by roscovitine. Half of the colonies were irradiated with UV (20J) (dotted), and the other half were used as control. Randomly chosen cells from each colony were used to start new genetically homogeneous colonies.

Figure 5.

Quantity and spectra of mutations in fibroblast colonies colonies identified by whole genome sequencing