Year of Publication

2002

Document Type

Dissertation

College

Graduate School

Department

Toxicology

First Advisor

Guo-Min Li

Second Advisor

Stephen Zimmer

Abstract

The stability of DNA is a critical factor for several diseases, the most prevalent of which is cancer. Several neurodegenerative and accelerated aging diseases are also characterized by genomic instability. The number and complexity of DNA repair pathways that human cells possess underscores the importance of genomic stability. These pathways ensure that damaged DNA is repaired and that a cells complement of DNA remains stable upon cell division. How one particular type of DNA alteration, a DNA loop, is processed in human cells was the focus of this study. We have employed an in vitro system to study defined DNA loop substrates by human nuclear extracts. The influence of either a 5 or 3 nick, the range of loop sizes processed, and the role of DNA mismatch repair, DNA nucleotide excision repair, and the Werner Syndrome helicase proteins were variables tested. The results indicate tha t DNA loops containing between 5 to 12 nucleotides are processed in a strand - specific manner when either a 5 or 3 nick is present , with repair being targeted solely to the nicked strand . This repair occurs by both mismatch repair dependent and independent pathways. The processing of DNA loops containing 30 nucleotides in length is directed either by a 5 nick, or by the loop itself, but not by a 3 nick. The nick independent pathway results solely in loop removal. The large loop pathway is independent of mismatch repair, nucleotide excision repair, and the WRN helicase/exonuclease protein. Both of the 5 nick directed pathways occur by excision that initiates at the pre- existing nick and proceeds towards the loop along the shortest path between the nick and loop. DNA resynthesis occurs using either DNA polymerase , , or and also initiates at the pre-existing 5 nick. The 3 nick directed intermediate loop repair pathway proceeds in a similar fashion, likely after a nick is made 5 to the loop region on the strand that contained the pre-existing nick. DNA synthesis inhibition has only a minor affect on the nick independent loop removal pathway as only a short tract of DNA surrounding the loop site is processed. In total, the results point to at least 3 novel pathways that process DNA loops that likely contribute to total genomic stability.

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