Structure-specific DNA binding and bipolar helicase activities of PcrA

Abstract

PcrA is an essential helicase in Gram-positive bacteria, but its precise role in cellular DNA metabolism is currently unknown. The Staphylococcus aureus PcrA helicase has both 5'-->3' and 3'-->5' helicase activities. In this work, we have studied the binding of S.aureus PcrA to a variety of DNA substrates that represent intermediates in DNA replication, repair, recombination and transcription. PcrA bound poorly or not at all to single-stranded DNA, double-stranded DNA with blunt ends, partially double-stranded DNA containing fork and bubble structures, and duplex DNA substrates containing either 5' or 3' single-stranded oligo dT tails. Interestingly, PcrA bound with high affinity to partially duplex DNA containing hairpin structures adjacent to a 6 nt long 5' single-stranded region and one unpaired nucleotide (flap) at the 3' end. However, PcrA did not detectably bind to partial duplexes with folded regions adjacent to a 6 nt long 3' single-stranded tail (with or without a 1 nt flap at the 5' end). PcrA showed moderate helicase activity with partially double-stranded DNAs containing 3' or 5' single-stranded oligo dT tails, the 3'-->5' helicase activity being more efficient than its 5'-->3' helicase activity. Interestingly, PcrA showed maximal helicase activity with substrates containing folded structures and 5' single-stranded tails, suggesting that its 5'-->3' helicase activity is greatly stimulated in the presence of specific structures. However, the 3'-->5' helicase activity of PcrA did not appear to be affected by the presence of folded substrates containing 3' single-stranded tails. Our data indicate that PcrA may recognize DNA substrates with specific structures in vivo and its 5'-->3' and 3'-->5' helicase activities may be involved in distinct cellular processes.

Figure 1

Structures of the various substrates used in this study.

Figure 2

Structure-specific DNA binding activity of the PcrA helicase. EMSAs were carried out using 5′ ³²P-labeled substrates (Table 1 and Figure 1) and the indicated amounts of PcrA. The DNA–protein complexes were resolved on polyacrylamide gels. P, probe; C, DNA–protein complex.

Figure 3

PcrA has bipolar 3′→5′ and 5′→3′ helicase activities. ³²P-labeled substrates were incubated with the indicated amounts of PcrA, and the products resolved by native PAGE. The probes used are listed in Table 1 and their structures are depicted in Figure 1. ds, substrates containing partial or fully duplex DNA; ss, single-stranded DNA.

Figure 4

PcrA efficiently unwinds structured substrates with 5′ ss regions. Various ³²P-labeled substrates containing a hairpin structure in their 5′ region were incubated with PcrA and subjected to EMSA.

Figure 5

Unwinding of DNA substrates containing hairpin structures at their 3′ regions by PcrA. ³²P-labeled substrates containing a hairpin structure in their 3′ region were incubated with PcrA and subjected to EMSA.