Molecular biology and enzymology of genetic recombination and DNA repair
Many classes of DNA rearrangements occur in all cells and play important roles in gene regulation, development, carcinogenesis, and evolution. The goal of this laboratory is to understand how these genetic rearrangements come about, and how they are regulated. To this end, we study a range of bacterial enzymes involved in recombinational DNA repair, both in vivo and in vitro. Part of the effort involves screens to identify relevant enzymes with new and sometimes unanticipated functions. In addition to understanding the enzymes themselves, we are increasingly exploring the intersection of recombination with other processes in DNA metabolism, and seeking an understanding of the factors that limit recombination within a cell.
There are currently seven projects underway in the laboratory:
1. The RecA protein. We have a longstanding interest in the bacterial RecA protein. This is the prototype of a family of recombinases that are present in organisms from bacteria to humans. Currently, RecA is at the center of our investigation of the limits of recombination. We are selecting for RecA protein variants with enhanced functionality, proteins that demonstrably increase rates of recombination during bacterial conjugation. The increased functionality is only one focus. The cost to the cell is another. Recombination is a double-edged sword, important when needed but potentially dangerous if it occurs at the wrong time and place. The variants with enhanced functionality often exhibit more persistent binding to DNA, making them impediments to DNA replication and transcription. Cells expressing the variants thus grow more slowly than those expressing the wild type protein. We wish to understand how other cellular processes impact the evolution of recombinase function.
2. The Ref protein, a programmable nuclease. The bacteriophage P1 encodes a protein called the recombination enhancement function or Ref. Over two decades ago, work in the laboratories of Max Gottesman and John Hayes showed that expression of this protein led to increases in the frequency of some recombination events in a RecA-dependent manner. Expecting to find a RecA regulator, we unexpectedly found that Ref was a novel RecA-dependent HNH family nuclease. Ref will only cleave DNA to which RecA is bound. If RecA protein is bound to an oligonucleotide, and used to form a D-loop in target DNA, Ref will cleave both strands of the target DNA within the D-loop. Ref, when deployed this way with RecA, is thus a CRISPR-like programmable nuclease that can be used to cut DNA at a desirable location. We are working hard to understand Ref function and mechanism of action. It is an unusual and fascinating little enzyme.
3. The MgsA/RarA protein. This protein is highly conserved from bacteria to humans, with important homologs in yeast (MGS1) and human (WRNIP1). Loss of this activity in any organism leads to a decline in genome stability. A few years ago, my student Asher Page, in collaboration with Jim Keck’s lab, solved the structure of this protein to provide the first structural information about this protein family. We are now actively engaged in determining the function of this protein, using approaches both in vivo and in vitro. It may have a novel function rescuing stalled replication forks.
4. The RadD (YejH) protein. The unstudied yejH gene was identified by Rose Byrne in the lab as a function needed for cells to recover from high doses of ionizing radiation. Stefanie Chen subsequently demonstrated that this gene encoded a protein needed in any situation involving DNA double strand breaks. Re-named RadD, we are exploring the function of this protein in vitro.
5. DNA polymerase V mechanism. DNA polymerase V is a specialized enzyme that can replicate DNA on damaged DNA templates. Induced during the bacterial SOS response, DNA polymerase V promotes mutagenic translesion synthesis (TLS). Activation of Pol V requires RecA protein at several steps, and a RecA monomer is a subunit of the active Pol V enzyme called pol V mut. Working with Myron Goodman at University of Southern California and Roger Woodgate at the NIH, we are slowly elucidating the unusual reaction cycle of this enzyme and the structural relationships between the various pol V mut subunits.
6. Resistance to extreme levels of ionizing radiation. Some bacteria, like Deinococcus radiodurans, display a robust capacity to survive exposure to astonishing levels of ionizing radiation (IR), sometimes a thousand times or more than doses that would be lethal to a human. To understand the factors most important in radiation resistance, we have carried out a directed evolution experiment, generating strains of E. coli that exhibit up to 10,000 fold greater survival after exposure to 3,000 Gy of IR. In one evolved population, we have been able to account for nearly the entire phenotype with three mutations in DNA repair genes: recA, dnaB, and yfjK. In other evolved populations, other strategies for radiation resistance may predominate. With this project, we are broadly exploring the molecular basis of extreme resistance to IR.
7. Forensic applications of double strand break repair. Each year, a small portion of the DNA samples submitted to crime laboratories cannot be analyzed due to degradation. The primary problem is DNA breaks that occur in samples that have been through a fire or explosion, or have simply been exposed too long to the elements. Using bacterial enzymes involved in double strand break repair (RecA, SSB, and DNA polymerase), we are developing a process to accurately repair double strand breaks in forensic DNA samples, to render more of these samples useful in crime investigations.
A broad approach to each of these problems is facilitated by active collaborations with over a dozen research groups around the world.