Biology subjects

Kimble, M. T.

Publications and source records attributed to Kimble, M. T..

5 recordsLinked to original sources

bioRxiv2025

Asymmetrical recognition and processing of double-strand breaks formed during DNA replication

DNA end resection to generate 3' ssDNA overhangs is the first step in homology-directed mechanisms of double-strand break (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 the size of an Okazaki fragment. We find that Mre11 preferentially binds blunt ends and is required for the removal Ku from these DSB ends. In contrast, the ends predicted to have 3' overhangs have minimal Ku binding, and end 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 reveals that Ku selectively binds nearly blunt ends, potentially explaining why replication-dependent DSBs are poorly repaired by non-homologous end joining. Significance StatementDNA end resection is a critical early step in the repair of DNA double-strand breaks by homologous recombination. Studies using endonucleases that generate DSBs in all phases of the cell cycle have shown that end resection proceeds by the sequential action of the Mre11 complex and long-range resection nucleases, Exo1 or Dna2-Sgs1. In this study, we restricted DSB generation to S-phase and found that the genetic requirements for end resection at more physiologically relevant DSBs are different to those at endonuclease-induced DSBs. The asymmetrical recognition and processing of replication-dependent DSBs may also explain the bias toward homologous recombination mediated repair over non-homologous end joining.

genetics↗

bioRxiv2024

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. We find that repair of replication-dependent DSBs requires homologous recombination (HR) and is independent of canonical non-homologous end joining. Consistent with a strong bias to repair these lesions using a sister chromatid template, we observe minimal induction of inter-chromosomal HR by nCas9. Using nCas9 and a gRNA to nick either the leading or lagging strand template, we carried out a genome-wide screen to identify factors necessary for the repair of replication-dependent DSBs. All the core HR genes were recovered in the screen with both gRNAs, but we recovered components of the replication-coupled nucleosome assembly (RCNA) pathway with only the gRNA targeting the leading strand template. By use of additional gRNAs, we find that the RCNA pathway is especially important to repair a leading strand fork collapse.

genetics↗

bioRxiv2022

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

Homologous recombination (HR), the error-free 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 Saccharomyces cerevisiae. This requirement for long-range resection is coupled to DNA damage checkpoint activation and the need for checkpoint proteins. Furthermore, artificial activation of the checkpoint partially restores interchromosomal recombination to exo1{Delta} sgs1{Delta} cells. However, cell cycle delay is insufficient to rescue the interchromosomal recombination defect of exo1{Delta} sgs1{Delta} cells, suggesting an additional role for the checkpoint. Given that the checkpoint is necessary for DNA damage-induced chromosome mobility, we propose that the importance of the checkpoint, and therefore long-range resection, in interchromosomal recombination is due to a need to increase chromosome mobility to facilitate pairing of distant sites. The need for long-range resection is circumvented when the DSB and its repair template are in close proximity.

genetics↗

bioRxiv2020

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 heavy (IgH) and light chains (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. In this approach, component Ig gene segments of the anti-PD1 antibody underwent de novo V(D)J recombination to diversify the anti-PD1 antibody in the primary antibody repertoire in the mouse models. Immunization of these mouse models further modifies the anti-PD1 antibodies through SHM. Known anti-PD1 antibodies block interaction of PD1 with its ligands to alleviate PD1-mediated T cell suppression, thereby boosting anti-tumor T cell responses. By diversifying one such anti-PD1 antibody, we derived many new anti-PD1 antibodies, including anti-PD1 antibodies with the opposite activity of enhancing PD1/ligand interaction. Such antibodies theoretically might suppress deleterious T cell activities in autoimmune diseases. The approach we describe should be generally applicable for diversifying other therapeutic antibodies. Significance StatementThe immune system is capable of producing enormous varieties of antibodies to counter pathogenic infections. In this study, we devised a method to harness the power of the immune system to produce potentially therapeutic antibodies. As a test, we used this method to generate antibodies against the human PD1 molecule, a negative regulator of T cell activity. Available anti-PD1 therapeutic antibodies inhibit the function of PD1, thereby boosting T cell activity against tumor cells. Through our approach, we diversified one such anti-PD1 antibody in our mouse models. From this work, we obtained multiple new anti-PD1 antibodies, some of which can stimulate, rather than inhibit, PD1 function in vitro. Such PD1 stimulatory antibodies could potentially be used to suppress pathogenic T cell activities in autoimmune diseases. This method could be used to diversify the functions of other therapeutic antibodies.

immunology↗

bioRxiv2020

Conditional Antibody Expression to Avoid Central B Cell Deletion in a 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 evolve from precursor antibodies through affinity maturation. To induce bnAbs, a vaccine must mediate a similar process of antibody maturation. One way to test vaccination strategies is to immunize mouse models that express human bnAb precursors. Such immunization experiments can assess whether the vaccine can convert precursor antibody into bnAb. A major problem with such mouse models is that bnAb expression often hinders B cell development in the bone marrow. Such developmental blocks may be attributed to unusual properties of bnAb variable regions, such as poly-reactivity and long antigen-binding loops, which are often under negative selection during primary B cell development. To address this problem, we devised a method to circumvent B cell developmental block by expressing bnAbs conditionally in mature B cells. We validated this method by expressing the unmutated common ancestor (UCA) of the human VRC26 bnAb in transgenic mice. Constitutive expression of combined immunoglobulin heavy and light chains of VRC26UCA led to developmental arrest of B cell progenitors in the bone marrow; poly-reactivity of VRC26UCA and poor pairing of VRC26UCA IgH chain with mouse surrogate light chain may contribute to the phenotype. The conditional expression strategy circumvented this developmental impediment, allowing the VRC26UCA to be expressed in mature peripheral B cells. This method should be generally applicable for expressing other antibodies that are under negative selection during B cell development. Significance statementMouse models can provide fast and cost-effective systems to test HIV-1 vaccine candidates at the pre-clinical stage. To serve this purpose, mouse models are engineered to express the precursors of human bnAbs against diverse HIV-1 strains. Immunization of such mouse models can evaluate the ability of vaccines to mature the precursor antibodies into bnAbs. However, due to unusual properties of bnAbs, mouse models expressing their precursors often have B cell developmental defects. In this study, we devised and validated a strategy to address this problem. This method could also facilitate the expression of other clinically relevant antibodies in mature B cells in transgenic mice; immunization of such mice could be used to generate novel antibodies with desirable properties.

immunology↗