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

• Main Author: Rye, Peter Thomas
• Other Authors: John M. Essigmann.
• Format: Others
• Language: English
• Published: Massachusetts Institute of Technology 2008
• Subjects: Chemistry.
• Online Access: http://dspace.mit.edu/handle/1721.1/36263; http://hdl.handle.net/1721.1/36263

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006. === Vita. === Includes bibliographical references. === 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. === (cont.) By monitoring the loss of radiosignal from a series of substrates that contained the label at prescribed distances downstream of the T:G mismatch, we were able to determine that VSPR patches are distributed around 2 to 4 deoxynucleotides in length. Interestingly, under certain reaction conditions, the addition of DNA Ligase improved the efficiency of repair initiation by Vsr, suggesting that VSPR may be optimal in the context of a multi-protein complex. Lastly, we investigated the effect of VSPR proteins on methyltransferase (MTase) repair of O6-methylguanine (6mG). MTase repair of O6mG opposite T results in a G:T mismatch that must be further processed to yield the native G:C base pairing. The G:T mismatch is therefore an intersection of the two pathways and led us to hypothesize that MTase and VSPR proteins might interact. Indeed, cells lacking the functions of MutS, MutL, or Vsr proteins displayed decreased MTase repair in vivo, revealing a previously unknown interaction. The cooperation between proteins of these two repair systems may shed light on the biological significance of the VSPR system. === by Peter Thomas Rye. === Ph.D.