Biochemistry and Molecular Biology

University of Texas Medical Branch


Faculty

Louise Prakash, Ph.D., Professor

One of the long-term objectives of the L. Prakash laboratory is to understand the means eukaryotic cells employ to replicate damaged DNA templates. A detailed understanding of the mechanisms of damage bypass could suggest ways to enhance the contribution of non-mutagenic bypass, thereby avoiding mutagenesis and carcinogenesis. The Prakash laboratory has been a pioneer in DNA repair studies in eukaryotes. 

Translesion Synthesis DNA polymerases of yeast and Humans: Genetic and biochemical studies in yeast carried out in the Prakash laboratory have indicated a major role for the Rad6-Rad18 protein complex in enabling replication through DNA lesions.Rad6, an ubiquitin conjugating enzyme, exists in vivo in a tight complex with Rad18, a DNA binding protein, and this protein complex controls the activation of three separate pathways of damage bypass, controlled by RAD5, RAD30, and REV3 genes, respectively.Rad5, a SWI/SNF DNA dependent ATPase, promotes the error-free bypass of DNA lesions, presumably by a "copy-choice" type of DNA synthesis.RAD30 encodes a DNA polymerase, Pol eta, which has the unique ability to replicate through UV induced cis-syn thymine-thymine dimers as proficiently and accurately as through undamaged DNA.Inactivation of Pol eta in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum.Pol eta is a low fidelity enzyme, relatively insensitive to geometric distortions in DNA, and able to replicate through a variety of DNA lesions.The mechanism of Pol eta action in lesion bypass is being analyzed using biochemical and structural studies.

Additionally, work from this group has defined the lesion bypass properties of various other eukaryotic DNA polymerases, including yeast Pol zeta, and human Pol kappa and Pol iota, and these studies have revealed a very high degree of specificity in their roles in lesion bypass.For example, Pol zeta is an efficient mismatch extender, and it promotes lesion bypass by extending from the nucleotides inserted opposite DNA lesions by another DNA polymerase.Pol kappa also has the ability to extend from mismatched base pairs on undamaged and damaged DNAs, but it is less efficient than Pol zeta at extending from nucleotides opposite DNA lesions.Pol iota functions in lesion bypass primarily by inserting nucleotides opposite DNA lesions.The mechanisms of lesion bypass by these polymerases are being studied by biochemical and structural means.

Other studies examine the role of ubiquitin conjugation in the various lesion bypass processes.In addition to the Rad6-Rad18 complex, which is required for all the lesion bypass processes, the Rad5-dependent "copy choice" mode of lesion bypass also requires the Mms2-Ubc13 ubiquitin-conjugating enzyme complex.Genetic and biochemical studies are in progress in yeast to define the role of these different ubiquitin conjugating enzyme complexes in promoting the exchange of the replicative polymerase by a translesion synthesis DNA polymerase at the lesion site or in promoting the other lesion bypass processes.