Repair of replication-dependent double-strand breaks differs between the leading and lagging strands.

Single-strand breaks (SSBs) are one of the most commonly occurring endogenous lesions with the potential to give rise to cytotoxic double-strand breaks (DSBs) during DNA replication. To investigate how replication-dependent DSBs are repaired, we employed Cas9 nickase (nCas9) to generate site- and strand-specific nicks in the budding yeast genome. We found that nCas9-induced nicks are converted to mostly double-ended DSBs during S phase.

Strand asymmetry in the repair of replication dependent double-strand breaks.

Single-strand breaks (SSBs) are one of the most common endogenous lesions and have the potential to give rise to cytotoxic double-strand breaks (DSBs) during DNA replication. To investigate the mechanism of replication fork collapse at SSBs and subsequent repair, we employed Cas9 nickase (nCas9) to generate site and strand-specific nicks in the budding yeast genome. We show that nCas9-induced nicks are converted to mostly double-ended DSBs during S-phase.

Genetic reporters to detect and quantify homologous recombination in yeast.

Homologous recombination is a conserved process that cells use to repair damaged DNA. Many genetic assays have been developed in Saccharomyces cerevisiae to measure and characterize different types of recombination events, as well as identify proteins acting in such recombination events. Here, we describe two intrachromosomal reporters that utilize ade2 heteroalleles, whereby homologous recombination can be detected by colony color and adenine prototrophy.

Long-range DNA end resection supports homologous recombination by checkpoint activation rather than extensive homology generation.

Homologous recombination (HR), the high-fidelity mechanism for double-strand break (DSB) repair, relies on DNA end resection by nucleolytic degradation of the 5'-terminated ends. However, the role of long-range resection mediated by Exo1 and/or Sgs1-Dna2 in HR is not fully understood. Here, we show that Exo1 and Sgs1 are dispensable for recombination between closely linked repeats, but are required for interchromosomal repeat recombination in .

An in vivo method for diversifying the functions of therapeutic antibodies.

V(D)J recombination generates mature B cells that express huge repertoires of primary antibodies as diverse immunoglobulin (Ig) heavy chain (IgH) and light chain (IgL) of their B cell antigen receptors (BCRs). Cognate antigen binding to BCR variable region domains activates B cells into the germinal center (GC) reaction in which somatic hypermutation (SHM) modifies primary variable region-encoding sequences, with subsequent selection for mutations that improve antigen-binding affinity, ultimately leading to antibody affinity maturation. Based on these principles, we developed a humanized mouse model approach to diversify an anti-PD1 therapeutic antibody and allow isolation of variants with novel properties.

Conditional antibody expression to avoid central B cell deletion in humanized HIV-1 vaccine mouse models.

HIV-1 vaccine development aims to elicit broadly neutralizing antibodies (bnAbs) against diverse viral strains. In some HIV-1-infected individuals, bnAbs evolved from precursor antibodies through affinity maturation. To induce bnAbs, a vaccine must mediate a similar antibody maturation process.

Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection.

The Mre11-Rad50-Xrs2 complex plays important roles in the DNA damage response by activating the Tel1 kinase and catalyzing 5'-3' resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5' strands at DSB ends in a reaction stimulated by Sae2 Accordingly, Mre11-nuclease deficient () and mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, cells exhibit greater sensitivity to genotoxins than cells.

Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires.

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120.