To investigate DNA replication enzymology across the nuclear genome of budding yeast, deep sequencing was used to establish the pattern of uncorrected replication errors generated by an asymmetric mutator variant of DNA polymerase δ (Pol δ). Sequencing of 16 genomes identified 1,206-bp substitutions generated over 33 generations by L612M Pol δ in a mismatch repair defective strain. Alignment of sequences flanking these substitutions identified "hotspot" motifs for Pol δ replication errors. The substitutions were distributed evenly across all 16 chromosomes. The vast majority were transitions that occurred with a strand bias that varied in a predictable manner relative to known functional origins of replication. This strand bias strongly supports the idea that Pol δ is primarily a lagging strand polymerase during replication across the entire nuclear genome.DNA polymerase δ | lagging strand replication | mutational hotspot | replication fidelity | mutator R eplication of the eukaryotic nuclear genome is intrinsically asymmetric, with a continuously replicated leading strand and a discontinuously replicated lagging strand (1). DNA polymerase α (Pol α) initiates new DNA chains and DNA polymerases ε (Pol ε) and δ (Pol δ), then performs the bulk of chain elongation. Variants of Pol ε and Pol δ (Pol δ L612M) that have distinctive error signatures were used to infer which DNA strand(s) each of these enzymes replicates in yeast. The results (2-4) are consistent with a model wherein Pol δ is primarily responsible for copying the lagging strand template, and Pol ε is primarily responsible for copying the leading strand template. Those studies used an 804-bp reporter gene adjacent to a single replication origin on chromosome 3 that fires frequently in early S phase (5). This situation is akin to "looking under a lamp post," because the yeast genome is 15,000 times larger (12 million bp, 16 chromosomes) and contains hundreds of replication origins that fire with different efficiencies and at various times in S phase (6). The genome also varies widely in sequence composition (7), and it is highly organized with respect to transcriptional status and chromatin content. Each of these variables may influence which of the many replication proteins are operating at replication forks, either directly or indirectly by affecting susceptibility to DNA damage. Among many questions about replication enzymology raised by the size and complexity of the nuclear genome, here we examine whether the role of Pol δ at the replication fork is constant or variable across the genome. To do so, we use deep sequencing to establish the pattern of base substitution mutations arising in a pol3-L612M mutant that is deficient in Msh2-dependent mismatch repair. Results and DiscussionRationale. To determine whether Pol δ primarily copies the lagging strand template across the whole genome, we made use of the mutational asymmetry of Pol δ L612M, which has high error rates for only two of the four possible mismatches that give rise to transitions (3,8). Thus...

Family X polymerases like DNA polymerase λ (pol λ) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5′ and 3′ ends of short gaps. DNA binding and gap filling are well characterized for one nucleotide gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that when bound to a two-nucleotide gap, pol λ scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket comprised of three conserved amino acids. Replacing these amino acids with alanine results in less processive gap filling and less efficient NHEJ involving two nucleotide gaps. Thus, akin to scrunching by RNA polymerase during transcription initiation, scrunching occurs during gap filling DNA synthesis associated with DNA repair.

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