2025 Theses Doctoral

Roles of the MRX Complex in the Repair of Replication-Dependent Double-Strand Breaks

Johnson, Matthew

In this project, we tell three stories detailing the role of the Mre11-Rad50-Xrs2 (MRX) complex in the repair of replication-dependent double strand breaks (DSBs). The MRX complex is one of the first protein complexes to be recruited to the site of a DSB and is implicated in promoting DNA end resection, tethering sister chromatids together, and ensuring error-free repair of DNA lesions.

Here, we explore the role of the MRX complex in regulating end resection at replication-dependent DSBs. DNA end resection to generate 3 ssDNA overhangs is the first step in homology-directed mechanisms of DSB repair. While end resection has been extensively studied in the repair of endonuclease-induced DSBs, little is known about how resection proceeds at DSBs generated during DNA replication. We previously established a system to generate replication-dependent double-ended DSBs at the sites of nicks induced by the Cas9D10A nickase in the budding yeast genome. Here, we suggest that these DSBs form in an asymmetric manner, with one break end being blunt or near blunt, and the other bearing a 3 ssDNA overhang of up to the size of an Okazaki fragment.

We find that Mre11 preferentially binds blunt ends and is required to evict Ku from these blunt DSB ends. In contrast, the ends predicted to have 3 overhangs have minimal Ku binding, and resection at these break ends can proceed in a mostly Mre11-independent manner through either the Exo1 or Dna2-Sgs1 long-range resection pathways. These findings indicate that resection proceeds differently at replication-dependent DSBs than at canonical DSBs, and reveal that Ku selectively binds nearly blunt ends, potentially explaining why replication-dependent DSBs are poorly repaired by non-homologous end joining. Next, we study the role the MRX complex plays in tethering sister chromatids together, and specifically focus our work on the Rad50 subunit. The Rad50 subunit of MRX has several poorly understood structural motifs that may be relevant for replication-dependent DSB repair. Several mutants have previously been generated in the RAD50 gene that perturb different domains of the protein including the coiled coil and hook domains. Structural predictions suggest that the physical sister chromatid tethering activity of the MRX complex would require cooperative action between the coiled-coil and hook domains of Rad50.

Here, we demonstrate that mutations in the coiled-coil domain are especially sensitive to the nickase, while hypomorphic hook domain mutants are modestly sensitive. This sensitivity is compounded by the loss of the sister chromatid tethering factor CTF8, suggesting a genetic interaction between MRX-mediated sister chromatid tethering and PCNA driven sister chromatid cohesion. To directly test whether mre11∆ and other nickase sensitive mutants have sister chromatid tethering defects, we have designed a fluorescent microscopy-based assay using a LacO/LacI-GFP construct within the same replicon as the Cas9D10A cut site.

Using this assay, we demonstrate that the MRX complex is required for sister chromatid tethering during S-phase after the induction of a replication-dependent DSB formed on the leading strand template. In contrast, we find that both long-range resection machinery and the Rad51 recombinase are dispensable for maintaining sister chromatid cohesion of replication-dependent DSBs during S-phase. Finally, we note that structural rad50 alleles confer a spontaneous defect in sister chromatid tethering, suggesting a role for the MRX complex in tethering outside of DSB repair.

Finally, we extend our study of DNA damage repair to study Cas9 nickase-induced mutagenesis and repair pathway choice. Cas9 nickases Cas9D10A and Cas9H840A, collectively termed nCas9, have been proposed as less mutagenic genome editors compared to canonical Cas9 given that they induce single-strand breaks (SSBs) instead of double-strand breaks (DSBs). However, there are relatively few studies that address the frequency and spectrum of nCas9-induced mutations. Using a classical genetic assay for loss of function, we demonstrate that nCas9 is indeed mutagenic, although much less so than canonical Cas9. nCas9-induced mutagenesis is mitigated by the MRX complex, while the translesion polymerase Rev3 contributes to SSB-induced mutations.

Furthermore, Exo1 also contributes to nCas9 mutagenesis, presumably by generating ssDNA that must be repaired via fill-in synthesis. In contrast to Cas9 DSBs, we find no contribution of the non-homologous end joining (NHEJ) pathway in nCas9 mutagenesis. Consistent with this finding, we demonstrate that while Cas9 inductions results in small indels at the site of the gRNA, nCas9 induction results in indels and base substitutions both at, as well as hundreds of nucleotides away from the gRNA locus. This finding highlights the need for sequencing regions surrounding the gRNA target site for gene editing applications using nCas9.