Biochemical characterization of the E. coli Very Short Patch Repair pathway and its coordination with methyltransferase repair of 0⁶-methylguanine

The E. coli Very Short Patch Repair (VSPR) system corrects T:G mismatches that arise through Dcm-mediated methylation and subsequent deamination of the underlined cytosine residue in the palindromic sequence 5'-CCWGG-3' (W is an adenine or thymine). Vsr initiates VSPR by producing a single stranded nick on the 5' side of the mismatched T. The MutS and MutL mismatch recognition proteins stimulate this activity, as cells lacking either of these proteins display diminished VSPR. Genetic studies also indicate that Pol I is responsible for removing and replacing a short tract of nucleotides downstream of the incision site and that DNA Ligase seals the nick to complete the repair event. However, until now, biochemical investigation of the repair steps downstream of Vsr incision have been lacking. Herein, we describe two novel in vitro assays used to probe the biochemical events of VSPR. The first was used to verify the reconstitution of VSPR using purified E. coli Vsr, Pol I, and DNA Ligase enzymes, while the second was used to measure the distribution of VSPR patch sizes in whole cell extracts.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2006

Author dc:creator

• Rye, Peter Thomas

Advisor dc:contributor.advisor

• John M. Essigmann.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/36263
• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Identifier URI

http://dspace.mit.edu/handle/1721.1/36263

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/36263