Asymmetric Regulation of Bipolar Single-stranded DNA Translocation by the Two Motors within Escherichia coli RecBCD Helicase

Fuqian Xie,Colin G Wu,Elizabeth Weiland,Timothy M Lohman

doi:10.1074/jbc.m112.423384

Abstract

Repair of double-stranded DNA breaks in Escherichia coli is initiated by the RecBCD helicase that possesses two superfamily-1 motors, RecB (3' to 5' translocase) and RecD (5' to 3' translocase), that operate on the complementary DNA strands to unwind duplex DNA. However, it is not known whether the RecB and RecD motors act independently or are functionally coupled. Here we show by directly monitoring ATP-driven single-stranded DNA translocation of RecBCD that the 5' to 3' rate is always faster than the 3' to 5' rate on DNA without a crossover hotspot instigator site and that the translocation rates are coupled asymmetrically. That is, RecB regulates both 3' to 5' and 5' to 3' translocation, whereas RecD only regulates 5' to 3' translocation. We show that the recently identified RecBC secondary translocase activity functions within RecBCD and that this contributes to the coupling. This coupling has implications for how RecBCD activity is regulated after it recognizes a crossover hotspot instigator sequence during DNA unwinding.

Highlights

RecBCD helicase is involved in repair of double-stranded DNA breaks
It has been shown that the RecD and RecB motors do not act concertedly within RecBCD [10, 13], it is not known whether the translocation activities of the two motors are independent or are functionally coupled
This is because the translocation rates of the individual RecB and RecD motors have not been measured within a functioning RecBCD holoenzyme but have only been inferred from DNA unwinding studies of RecBCD and variants possessing mutations within the RecB and RecD motors by assuming motor independence [10, 16, 18]

Summary

Background

RecBCD helicase is involved in repair of double-stranded DNA breaks. Results: The 5Ј to 3Ј ssDNA translocation rate of RecBCD is faster than the 3Ј to 5Ј rate in the absence of a CHI site, and the rates are coupled asymmetrically. Repair of double-stranded DNA breaks in Escherichia coli is initiated by the RecBCD helicase that possesses two superfamily-1 motors, RecB (3؅ to 5؅ translocase) and RecD (5؅ to 3؅ translocase), that operate on the complementary DNA strands to unwind duplex DNA. It is not known whether the RecB and RecD motors act independently or are functionally coupled. We show by directly monitoring ATP-driven single-stranded DNA translocation of RecBCD that the 5؅ to 3؅ rate is always faster than the 3؅ to 5؅ rate on DNA without a crossover hotspot instigator site and that the translocation rates are coupled asymmetrically.

To whom correspondence should be addressed

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION