UvrABC incision of N-methylmitomycin A-DNA monoadducts and cross-links

J Biol Chem. 1989 Dec 5;264(34):20697-704.


The Escherichia coli UvrABC endonuclease is a multisubunit enzyme that initiates the repair of a wide variety of DNA lesions in vivo by making dual incisions on a damaged strand at the eighth or ninth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified base. It has been hypothesized that UvrABC is able to recognize a broad spectrum of lesions because it does not recognize the lesion per se but rather gross helical distortions that the lesion induces in the DNA. Several lesions have recently been studied which are thermal stabilizing and are not believed to distort the DNA grossly, including the CC-1065-N-3-adenine and anthramycin-N-2-guanine adducts. We have studied the activity of UvrABC in vitro on another thermal stabilizing and nondistortive adduct, N-methylmitomycin A (NMA), a bifunctional DNA-alkylating agent that reacts with guanine on the side facing the minor groove, yielding either monoadducts or interstrand cross-links. NMA adducts increase the thermal stability of DNA, and theoretical calculations indicate that NMA adducts do not grossly distort the DNA helix. Our results show that UvrABC makes incisions at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to an NMA monoadduct, consistent with the incision pattern observed for the majority of other lesions that are also recognized by UvrABC. DNA containing a site-specific NMA cross-link was also recognized and incised by UvrABC. The rate of incision of NMA cross-linked DNA was about 200-fold higher in supercoiled molecules than in relaxed molecules, whereas the rate of incision of DNA containing NMA monoadducts was stimulated approximately 2-fold by supercoiling. The signal for UvrABC recognition and incision of damaged DNA is discussed in relation to the ability of UvrABC to incise NMA adducts as well as other nondistortive lesions.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Cross-Linking Reagents / pharmacology*
  • DNA / metabolism*
  • DNA, Circular / metabolism
  • Endodeoxyribonucleases / metabolism*
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins*
  • Kinetics
  • Mitomycins / pharmacology*
  • Molecular Sequence Data
  • Plasmids
  • Restriction Mapping
  • Substrate Specificity


  • Cross-Linking Reagents
  • DNA, Circular
  • Escherichia coli Proteins
  • Mitomycins
  • DNA
  • Endodeoxyribonucleases
  • endodeoxyribonuclease uvrABC
  • N-methylmitomycin A