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, 284 (3), 1523-32

Initiation of New DNA Strands by the Herpes Simplex virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase Alpha

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Initiation of New DNA Strands by the Herpes Simplex virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase Alpha

Nisha A Cavanaugh et al. J Biol Chem.

Abstract

A key set of reactions for the initiation of new DNA strands during herpes simplex virus-1 replication consists of the primase-catalyzed synthesis of short RNA primers followed by polymerase-catalyzed DNA synthesis (i.e. primase-coupled polymerase activity). Herpes primase (UL5-UL52-UL8) synthesizes products from 2 to approximately 13 nucleotides long. However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at least 8 nucleotides long. Surprisingly, coupled activity was remarkably inefficient, even considering only those primers at least 8 nucleotides long, and herpes polymerase typically elongated <2% of the primase-synthesized primers. Of those primers elongated, only 4-26% of the primers were passed directly from the primase to the polymerase (UL30-UL42) without dissociating into solution. Comparing RNA primer-templates and DNA primer-templates of identical sequence showed that herpes polymerase greatly preferred to elongate the DNA primer by 650-26,000-fold, thus accounting for the extremely low efficiency with which herpes polymerase elongated primase-synthesized primers. Curiously, one of the DNA polymerases of the host cell, polymerase alpha (p70-p180 or p49-p58-p70-p180 complex), extended herpes primase-synthesized RNA primers much more efficiently than the viral polymerase, raising the possibility that the viral polymerase may not be the only one involved in herpes DNA replication.

Figures

FIGURE 1.
FIGURE 1.
Herpes primase and primase-coupled polymerase activities on 3′-d(T20GTCCT36)-5′. A, products of primase-coupled polymerase assays were measured in assays containing enzymes as noted, and either [α-32P]GTP or [α-32P]dATP as noted, and were performed as described under “Experimental Procedures.” Product lengths (shown) were determined using 5′-[32P]dA40 as a marker, as well as pppApG and pppApGpA synthesized using human primase, ATP, and [α-32P]GTP. B, length of the RNA primers elongated by the polymerase during primase-coupled polymerase activity was determined in assays containing primase-helicase, 3′-d(T20GTCCT36)-5′, NTPs, [α-32P]dNTPs, and either herpes polymerase (UL30-UL42) or human pol α. Lane 1 shows the coupled products. A small fraction of the coupled products were gel-extracted (lane 2) and treated with binase (lane 3) as described under “Experimental Procedures.” Product lengths (shown) for herpes polymerase-herpes primase-coupled activity were determined using 5′-[32P]dA40 as a marker. Product lengths (shown) for pol α-herpes primase coupled activity were estimated using 5′-[32P]dC30. In this, and subsequent gel images, sections taken from different parts of a larger gel are separated. nts, nucleotides.
FIGURE 2.
FIGURE 2.
Effects of increasing the dNTP concentration on primase-coupled polymerase activity. Assays contained primase-helicase, polymerase (UL30-UL42), 3′-d(T20GTCCT36)-5′, and either [α-32P]NTPs or NTPs and [α-32P]dNTPs to measure primase activity and primase-coupled polymerase activity, respectively. Coupled activity was measured in terms of pmol of dATP incorporated. The fraction of primers elongated was determined as described under “Experimental Procedures.”
SCHEME 1.
SCHEME 1.
Differentiating intramolecular and intermolecular primer transfer.
FIGURE 3.
FIGURE 3.
Mechanism of primer transfer during primase-coupled polymerase activity. Assays were performed as described under “Experimental Procedures” and contained the template 3′-d(T20GTCCT36)-5′, NTPs, [α-32P]dNTPs, and the noted enzymes. Product lengths (shown) were determined as described in Fig. 1A. The spots present in the lanes, including binase, are most likely a result from binase modifying a product generated by the interaction of primase with either [α-32P]dNTPs or with a radiolabeled contaminant in the [α-32P]dNTPs (2nd lane), because they do not appear in the binase-only lane (1st lane).
FIGURE 4.
FIGURE 4.
UL8 stimulates primase-coupled polymerase activity with the core primase-helicase (UL5-UL52). Primase-coupled polymerase assays contained 3′-d(T20GTCCT36)-5′, either UL5-UL52-UL8 or UL5-UL52, and also contained other proteins (UL8, UL30-UL42, and binase) as noted. A, relative primase activity using [α-32P]NTPs. B, primase-coupled polymerase activity using [α-32P]dNTPs. Product lengths (shown) were determined using 5′-[32P]dA40 and 5′-[32P]dC30 as markers.
FIGURE 5.
FIGURE 5.
Primase-coupled polymerase activity between herpes primase and human pol α. Assays were performed as described under “Experimental Procedures” and contained the template 3′-d(T20GTCCT36)-5′, NTPs, [α-32P]dNTPs, and the noted enzymes. Product lengths (shown) were estimated using 5′-[32P]dA40 and 5′-[32P]dC30 as markers.

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