Abstract
Closely opposed lesions form a unique class of DNA damage that is generated by ionizing radiation. Improper repair of closely opposed lesions could lead to the formation of double strand breaks that can result in increased lethality and mutagenesis. In vitro processing of closely opposed lesions was studied using double-stranded DNA containing a nick in close proximity opposite to a dihydrouracil. In this study we showed that HU protein, an Escherichia coli DNA-binding protein, has a role in the repair of closely opposed lesions. The repair of dihydrouracil is initiated by E. coli endonuclease III and processed via the base excision repair pathway. HU protein was shown to inhibit the rate of removal of dihydrouracil by endonuclease III only when the DNA substrate contained a nick in close proximity opposite to the dihydrouracil. In contrast, HU protein did not inhibit the subsequent steps of the base excision repair pathway, namely the DNA synthesis and ligation reactions catalyzed by E. coli DNA polymerase and E. coli DNA ligase, respectively. The nick-dependent selective inhibition of endonuclease III activity by HU protein suggests that HU could play a role in reducing the formation of double strand breaks in E. coli.
Highlights
Ionizing radiation generates a wide spectrum of DNA damage, including strand breaks, abasic (AP)1 sites, base damages, and cross-links [1,2]
HU protein did not inhibit the subsequent steps of the base excision repair pathway, namely the DNA synthesis and ligation reactions catalyzed by E. coli DNA polymerase and E. coli DNA ligase, respectively
When duplex L was used as a substrate, increasing the amount of HU protein had no effect on the activity of E. coli endonuclease III (Fig. 1, panel A)
Summary
HU could potentially mediate the sequential repair of a clustered lesion consisting of closely opposed DNA damage. These data further suggest that DNA-binding proteins that have high affinity for nicks, such as poly (ADP-ribose) DNA polymerase, could mediate similar processes in the cells
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