Nelson:Projects

Bacteriophage T4 Recombination-dependent DNA Repair

T4 phage is able to perform all the functions required for the replication and maintenance of its genome. Decades of research has proven T4 phage to be an excellent model system. The molecular mechanisms of the core DNA replication proteins (e.g., polymerase, helicase, clamp protein) are well conserved from phage to humans. It is expected that this conservation will extend to the DNA repair enzymes.

Mre11/Rad50:

Mre11 and Rad50 contain nuclease and DNA-dependent ATPase activities, respectively, and are known to be involved in the repair of double strand breaks (DSB. Based on in vivo experiments, it is thought that these proteins are responsible for the 5’→3’ resection of a DSB. However, the in vitro exonuclease activity of Mre11 is not compatible with this role. This has led to a controversy in the field with regard to the true function of the Mre11/Rad50 complex. Until recently, the Mre11/Rad50 homologs from T4 phage had never been successfully expressed and purified. We have developed methods to achieve mg quantities of highly purified enzymes. This availability, along with the large amount of genetic data from the T4 system, makes T4 Mre11 and Rad50 excellent candidates for investigation into the molecular mechanisms of what has been called the “keystone complex” of DNA repair.

UvsW Helicase:

UvsW helicase is one of three helicases found in T4 phage and is a member of the sf2 family of proteins. The sf2 proteins are grouped according to sequence homology and are ATP-dependent motor proteins that translocate on either RNA or DNA templates. Many of the sf2 proteins are helicases, however some have only translocation ability and are thought to be involved in the remodeling of their nucleic acid substrates. We recently discovered that UvsW has both strand annealing and duplex unwinding activities. Strand annealing is not due to simple microscopic reversibility (i.e., strand annealing is not the chemical reversal of duplex unwinding). UvsW is the only characterized non-human protein that contains both strand annealing and duplex unwinding activities in a single polypeptide chain. The human proteins with these activities are Werner’s syndrome helicase (WRN) and Bloom’s syndrome helicase (BLM. Recent work has suggested that both activities are necessary for the remodeling of stalled replication forks and bypass of DNA lesions by the replication machinery. WRN and BLM helicases are composed of 1432 and 1417 amino acids, respectively, as compared to 502 for UvsW. This drastic size difference is due to the presence of several additional protein binding domains in the human enzymes. It is very likely that UvsW and WRN/BLM share the same basic molecular mechanisms since they have identical substrate specificity and similar functional properties.

Mammalian Mitochondrial DNA Replication

The necessity for DNA repair in mitochondria was recognized over 50 years ago when the free radical theory was proposed by Harman. It was hypothesized that the aging process is due to the cumulative build up of oxidative damage in mitochondrial DNA (mtDNA). Since then, a great deal of evidence has been found in support of this hypothesis. Additionally, links have been found between damaged mtDNA and diseases such as Leber’s hereditary optic neuropathy, chronic progressive external opthalmoplegia, Alzheimer’s, Parkinson’s, and cancer. The most extensively studied mitochondrial DNA repair mechanism is base excision repair. Mitochondria from several organisms contain DNA repair enzymes such as glycosylase, AP endonuclease and DNA ligase III. The mechanisms for double-strand break (DSB) repair are much less characterized. DSBs are arguably the most drastic type of DNA damage and are repaired through recombination or non-homologous end joining. Analysis of mtDNA rearrangements has indicated that homologous recombination occurs in mitochondria from fungi, plants, and animals. However, the proteins and enzymes that carry out this process have not been isolated.

Replicative Helicase (twinkle):

Polymerase gamma A:

Polymerase gamma B:

Single-stranded DNA Binding Protein:

Topoisomerase:

RNA polymerase (primase):