Inhibition of translesion DNA polymerase by archaeal reverse gyrase

Abstract

Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.

The Archaeal Topoisomerase Reverse Gyrase Is a Helix-destabilizing Protein That Unwinds Four-way DNA Junctions

“…4B), which impairs both DNA relaxation and positive supercoiling (19). Surprisingly, the Y965F protein was able to process HJ efficiently, although as expected, no cleavage products were obtained (Fig.…”

“…Both RG and its isolated N-terminal domain show DNA-dependent ATPase activity, which is essential for the positive supercoiling reaction (19,21); however, HJ processing by RG was unaffected by the addition of 5 mM ATP (Fig. 4A), 0.5 mM MgCl 2 , which is required for ATPase activity, or 1 mM EDTA, which inhibits ATPase activity (data not shown).…”

“…Proteins were eluted with a linear gradient of NaCl (0 -1 M NaCl). Throughout purification, fractions were analyzed by SDS-PAGE and Western blotting using the anti-RG polyclonal antibody (19). Positive fractions were pooled, concentrated to a final volume of 2 ml, dialyzed against phenyl buffer (50 mM phosphate buffer pH.…”

“…Western Blots-Western blots were performed as described previously (19) using the anti-RG polyclonal antibody, which recognizes both recombinant TopR1 and recombinant TopR2 3 as well as reverse gyrase in S. solfataricus cell extracts (19).…”

“…Several results suggest that reverse gyrase might participate in the cell response to DNA damage. In the crenarchaeon Sulfolobus solfataricus, reverse gyrase is recruited to DNA after UV irradiation (17), interacts with the single strand-binding protein (SSB), and the translesion DNA polymerase, PolY (18,19), and is degraded after treatment with methyl methane sulfonate (MMS) (20). Reverse gyrase structure and its involvement in genome stability are reminiscent of the evolutionary conserved complexes comprising topoisomerase III and members of the RecQ helicase family, which have essential functions in recombination and repair (for a recent review, see Ref.…”