20.109(F17):Test role of biochemical factors in genomic stability (Day4)

Introduction

In the initial experiment for this module, your goal was to optimize the CometChip assay. Now that you have data concerning how to best load cells into the CometChip, you will use this technique to study the Base Excision Repair (BER) pathway. BER corrects DNA damage, specifically base lesions, that result from oxidation, alkylation, deamination, and depuriniation/depryrimidination. If base lesions are not repaired, non-canonical base pairing can occur, which may result in the incorporation of an incorrect base during replication. To prevent mutations and maintain integrity of the genome, the BER pathway evolved as a highly conserved repair mechanism in both E. coli and mammals.

Base excision repair pathway.

The core BER pathway includes only four proteins that function to remove the damaged base and replace it with the correct base. First, a DNA glycosylase recognizes that a damaged base is present in the DNA and cleaves an N-glycosidic bond, which creates an apurinic or apyrimidinic site (referred to as an AP site in both cases). Different DNA glycosylases recognize different types of base lesions. Second, the DNA backbone is cleaved to create a single-strand DNA nick by either a DNA AP endonuclease or a DNA AP lyase. Next, a DNA polymerase incorporates the correct base using the sister strand as a template. Last, a DNA ligase completes the repair by sealing the single-strand nick, which restores integrity to the helix. For a more detailed description of the BER pathway, read this review by Robertson et al.

The BER pathway repairs base lesions using either a short-patch or long-patch mechanism. In the short-patch BER pathway, a single nucleotide is removed in the repair process. In contrast, in the long-patch BER pathway at least two nucleotides are removed. Though both mechanisms of repair are utilized, the conditions that drive each are poorly understood.

To study the BER pathway, as a class you will use two chemical treatments to generate base lesions. The treatments will be applied to two CometChips as a second variable will be introduced during the next laboratory session.

Protocols

Reagents list

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