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Comparative Study
. 1999 Aug 3;96(16):9045-50.
doi: 10.1073/pnas.96.16.9045.

A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil

M A Greagg  1 , M J Fogg, G Panayotou, S J Evans, B A Connolly, L H Pearl
Affiliations
Comparative Study

A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil

M A Greagg et al. Proc Natl Acad Sci U S A. .

Abstract

Deamination of cytosine to uracil is the most common promutagenic change in DNA, and it is greatly increased at the elevated growth temperatures of hyperthermophilic archaea. If not repaired to cytosine prior to replication, uracil in a template strand directs incorporation of adenine, generating a G.C --> A.U transition mutation in half the progeny. Surprisingly, genomic analysis of archaea has so far failed to reveal any homologues of either of the known families of uracil-DNA glycosylases responsible for initiating the base-excision repair of uracil in DNA, which is otherwise universal. Here we show that DNA polymerases from several hyperthermophilic archaea (including Vent and Pfu) specifically recognize the presence of uracil in a template strand and stall DNA synthesis before mutagenic misincorporation of adenine. A specific template-checking function in a DNA polymerase has not been observed previously, and it may represent the first step in a pathway for the repair of cytosine deamination in archaea.

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Figures

Figure 1
Figure 1
Long-range primer extension reactions on normal and single-uracil templates. (a) Denaturing polyacrylamide gel showing products of reactions in which a 31-mer primer was extended against a 119-nucleotide template (see Methods) containing either a deoxyuridine (U) or a deoxythymidine (T) 23 nucleotides from the 5′ end. Reactions were performed with DNA polymerases from Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo), and Thermus aquaticus (Taq). All three polymerases produce full-length products on T templates, but the archaeal enzymes produce smaller major products on U templates. (b) Polyacrylamide gel showing the position of the major premature termination product from the primer extension reaction against the U template with Pyrococcus furiosus DNA polymerase (Pfu exo+), relative to a sequencing ladder of the segment of pUC19 used in the long-range primer extension reaction (see Methods). The sequence for the template strand is given, reading 3′→5′ from the bottom (shorter primer extension products) to the top (longer primer extension products). The position of the deoxyuridine (U) is indicated. The major Pfu products correspond to termination of the polymerase reaction 4–6 bases upstream of the template deoxyuridine. The position of the full-length product obtained from primer-extension against the T template (not shown) was consistent with termination at the end of the 119-base template.
Figure 2
Figure 2
Short-range primer extensions. Denaturing polyacrylamide gels showing short-range primer extension products on otherwise identical templates containing either no uracil (no dU) or a single uracil positioned 1–7 bases (dU+n) from the end of the primer-template duplex region. The first lane (primer) contains just the primer strand as a marker. Gels are for the following: Vent exo+ and exo− (a), Pfu exo+ and exo− (b), Pwo exo+ (c), Taq (d), and T4 (e). In all cases the “no dU” lane shows the maximum-length 44-mer product. All the archaeal enzymes fail to produce full-length product on templates containing uracil, but they are able to extend the primer slightly when the uracil is 5 or more bases beyond the end of the duplex region. Products shorter than the original primer are present in reactions involving enzymes with functional 3′→5′ exonuclease activity (exo+).
Figure 3
Figure 3
Time course of short-range primer extension and effect of an abasic site in the template. (a) Denaturing polyacrylamide gels showing the short-range primer extension products produced by Pfu (exo−) on a template containing a single uracil positioned 10 bases from the end of the primer-template duplex region. The time of the incubation was varied as shown and in all cases a truncated product is produced. The first lane (primer) contains just the primer strand as a marker. The second lane (no dU) shows the maximum length 44-mer product. (b) Denaturing polyacrylamide gels showing the short-range primer extension products produced by Pfu (exo−) on a template containing either a stable abasic site (Ab+10) or a single uracil (dU+10) positioned 10 bases from the end of the primer-template duplex region. The first two lanes contain markers 24 (primer size) and 34 bases in length. With Ab+10 two terminated products, 35 and 36 bases long, are produced. These correspond to incorporation of a dNTP at the abasic site followed by the random non-template-directed addition of two further nucleotides. As the polymerase used lacks a functional 3′-5′ exonuclease, these nontemplate additions persist, so that the full-length product is longer than the 44-mer (marker, and small amount of full-length 44-mer produced in the dU+10 case). With the dU+10 template, the terminated product is shorter, clearly indicating a stop preceding the dU rather than in the vicinity of the dU.
Figure 4
Figure 4
DNA-binding specificity of Pfu DNA polymerase. (a) Surface plasmon resonance measurements of Pfu DNA polymerase interactions with DNA. The signal in response units (RU) is proportional to the mass of enzyme bound to the oligonucleotides immobilized on the surface of the Biacore chip. The beginning and end of injection of Pfu DNA polymerase over the chip surface are indicated by the arrows. Interactions were measured with single-stranded 35-mer oligonucleotides containing either a single uracil (ssU), a single abasic site (ssab), or no modification (ssC), and with double-stranded 35-mers containing a single U⋅G mispair (U:G) or a guanine opposite and abasic site (ab:G). Pfu polymerase interacts strongly with ssU but no other oligonucleotides. (b) Dose–response curve for binding of Pfu polymerase to ssU. Experimental data (○) were fit to the equation R = Rmax⋅C/(Kd + C), where C is the protein concentration and Rmax is the maximal response, giving an estimated Kd = 0.54 μM.
Figure 5
Figure 5
Read-ahead uracil detector. (a) Blocking of a running polymerase actively extending a primer, by a second polymerase bound to deoxyuridine in the single-stranded segment of the template, would give a gap between the last incorporated nucleotide (arrowed) and the template deoxyuridine (U), larger than the “footprint” of the polymerase. (b) In the read-ahead model, specific stalling of a running polymerase at a deoxyuridine upstream in the single-stranded segment of the template would give a gap between the last incorporated nucleotide (arrowed) and the template deoxyuridine (U), smaller than the “footprint” of the polymerase, as is observed.
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