Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

Abstract

UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3 to 5 direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNAprotein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the crosslink-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent (ϳ70°), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links.

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

“…It has been shown that DPCs impede the progression of the UvrD helicase involved in nucleotide excision repair only when placed on the translocating strand (14). The progression of T7 RNA polymerase that transiently disrupts dsDNA for transcription is also blocked by DPCs only when DPCs are placed on the transcribed strand (17).…”

Functions that protect Escherichia coli from DNA–protein crosslinks

“…The DinG, UvrD, and Rep helicases have been implicated in preventing or mitigating the damage from collisions between the replication machinery and bound proteins (such as RNA polymerase), and at least Rep and UvrD have protein removal activity in vitro (13,18–20). Specific to blocked transcription complexes, Mfd, the transcription-coupled repair factor in bacteria, recognizes RNAP stalled at DNA damage such as a pyrimidine dimer, removes RNAP from the DNA, and recruits excision repair machinery (21,22).…”

T7 RNA Polymerases Backed up by Covalently Trapped Proteins Catalyze Highly Error Prone Transcription

“…With respect to repair, DPCs impair the loading of UvrB, a damagerecognition protein involved in bacterial nucleotide excision repair (NER), onto the DPC site in a DPC size-dependent manner (7). The progression of the bacterial NER helicase (UvrD) is severely blocked when the DPC is present in the translocating strand (8). In mammalian cells the upper size limit of crosslinked proteins (CLPs) amenable to NER is around 8 kDa, eliminating the role of NER in the repair of DPCs in vivo (9).…”