Influence of DNA End Structure on the Mechanism of Initiation of DNA Unwinding by the E. coli RecBCD and RecBC Helicases

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Abstract E. coli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3' to 5' translocase and RecD, a 5' to 3' translocase), that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can " melt-out " ~ six bp upon binding to the end of a blunt-ended DNA duplex in a Mg 2+-dependent but ATP-independent reaction, we have investigated the effects of non-complementary single-stranded (ss) DNA tails (3'-(dT) 6 and 5'-(dT) 6 or 5'-(dT) 10) on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3'-(dT) 6 and 5'-(dT) 6 non-complementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk U = 774 ± 16 bp s-1) and kinetic step-size (m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k C = 45 ± 2 s-1). However, when the non-complementary 5'-ssDNA tail is extended to ten nucleotides (5'-(dT) 10 and 3'-(dT) 6), the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk U = 538 ± 24 bp s-1) is observed with a similar kinetic step-size (m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing non-complementary twin 5'-dT 6 and 3'-dT 6 tails, and unwinding can be described by a simple uniform n-step ACCEPTED MANUSCRIPT 3 sequential scheme, without the need of the additional k …