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Eukaryotic DNA Advanced article Polymerases Article Contents . Introduction Sue Cotterill, St George’s Hospital Medical School, London, UK . Structural Diversity of Eukaryotic Cellular Polymerases . Polymerases Involved in Chromosome Replication Stephen Kearsey, Department of Zoology, University of Oxford, Oxford, UK . Factors Involved in Polymerase Function at the Repli- cation Fork . Polymerases with Specialized Functions in Genome Replication . Polymerases Involved in DNA Repair . Polymerases Involved in Base Excision Repair . Translesion Polymerases . Polymerases Involved in NHEJ . Polymerase with Unknown Functions . Catalytic Function of Polymerases . Assays . Current Research Topics and Unanswered Questions Online posting date: 15th December 2009 Deoxyribonucleic acid (DNA) is replicated and repaired by Introduction a family of enzymes called DNA polymerases. Eukaryotic cells have a diversity of these enzymes that, while sharing The major function of deoxyribonucleic acid (DNA) a common biochemical activity, are specialized for par- polymerases is to replicate the genome and thus to allow ticular roles. Three polymerases are required for the rep- transmission of genetic information from one generation lication of the nuclear genome, with pol a involved in to the next. In eukaryotic cells, this takes place during a priming and initial synthesis and pols d and e involved in discrete period (S phase) of interphase, and replicated bulk DNA replication. These polymerases are dependent chromosomes are subsequently segregated to daughter cells during mitosis. As well as replicating DNA, poly- on a large number of other proteins which unwind the merases help to maintain the integrity of the genome by DNA and perform other functions essential for efficient participating in various modes of DNA repair. DNA DNA synthesis. Polymerases are also involved in DNA polymerases are also required for the replication of repair and many repair-specific enzymes have been iden- eukaryotic viruses. Some viruses, such as SV40, use host tified. Some repair polymerases can refill a gap generated polymerases while others, such as adenovirus, encode their by removal of damaged DNA, or copy a damaged own polymerase; discussion of viral polymerases is outside template, allowing DNA synthesis to proceed across a the scope of this review. See also: Cell Cycle damaged template. Repair polymerases can also have The basic catalytic reaction of DNA polymerases is to tissue-specific functions in lymphoid cells, where they effect semiconservative replication of DNA, using a single- contribute to somatic hypermutation of immunoglobulin stranded DNA chain as a template and four deoxynucleo- genes. tides (deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine triphosphate (dATP)) as precursors for DNA synthesis (Figure 1). The enzyme assembles the pre- cursor nucleotides on the template to form a complementary DNA strand, selecting the incoming nucleotide using the base ELS subject area: Biochemistry pair rules A . TandG. C. To start synthesis on a single- stranded DNA molecule, DNA polymerases need a primer. How to cite: This is a length of ribonucleic acid (RNA) or DNA that is Cotterill, Sue; and Kearsey, Stephen (December 2009) Eukaryotic DNA annealed to the single-stranded template. The primer pro- Polymerases. In: Encyclopedia of Life Sciences (ELS). John Wiley & Sons, vides a 3’-OH that can be extended by the polymerase; this Ltd: Chichester. configuration of the primer is important because polymerases DOI: 10.1002/9780470015902.a0001045.pub2 can only extend a new chain in the 5’ to 3’ direction. If the ENCYCLOPEDIA OF LIFE SCIENCES & 2009, John Wiley & Sons, Ltd. www.els.net 1 Eukaryotic DNA Polymerases Primer 5’ – A T G – 3’ OH Table 1 Eukaryotic DNA polymerases Template 3’ – T A CGATT Polymerase Family Function +dCTP y (theta) A Base-excision repair? Primer 5’ – A T GC – 3’ OH n (nu) A ? Template 3’ – T A CGATT g (gamma) A Mitochondrial DNA replication (a) +PPi a (alpha) B Chromosome replication (initiation, Okazaki frag- +dNTP ment priming) Polymerase dNTP Polymerase Polymerase Polymerase Template Template Template d (delta) B Chromosome replication Template Nx Nx+1 Nx (elongation, lagging strand (b) +PPi synthesis) Figure 1 (a) Basic mechanism catalysed by DNA polymerases. Nucleotide excision repair, Polymerization of nucleotides on the single-stranded template requires a mismatch repair primer (RNA or DNA) which provides a 3’-OH group to which the incoming e (epsilon) B Chromosome replication nucleotide is joined and the direction of synthesis is thus 5’ to 3’.Only (elongation, leading strand nucleotides that correctly base pair with the templates strand (according to synthesis) A . T, G . C rules) are incorporated. One molecule of pyrophosphate (PPi) is produced per nucleotide incorporated. (b) Steps in the polymerization Nucleotide excision repair, reaction. The order of the reaction is binding to the template–primer, mismatch repair? followed by binding of the dNTP. dNTP is cleaved at the a/b bond to give z (zeta) B Translesion synthesis dNMP that is added onto the chain, PP is released, and the polymerase i (beta) X Base-excision repair translocates along to the next 3’ terminus or dissociates. b l (lambda) X Base-excision repair, non- homologous end joining m (mu) X Nonhomologous end DNA template to be replicated is double-stranded, poly- joining merase action still needs a primer and, in addition, re- TdT X Nonhomologous end quires other enzymes to unwind the double helix. See also: joining DNA Helicases; DNA Polymerase Fidelity Mechanisms; Z (eta) Y Translesion synthesis, Polymerase Processivity: Measurement and Mechanisms error-free replication of CPDs i (iota) Y Translesion synthesis k (kappa) Y Translesion synthesis, Structural Diversity of Eukaryotic Cel- nucleotide-excision repair lular Polymerases Rev1 Y Translesion synthesis Telomerase RT Telomere maintenance Although all DNA polymerases share the same basic DNA polymerase s (Trf4/5), originally implicated in chromosome catalytic mechanism, at least 16 distinct polymerases have cohesion (Wang et al., 2000), is in fact a poly(A) RNA polymerase and been identified in human cells (Hubscher et al., 2002; has no DNA polymerase activity (Haracska et al., 2005). Trf4 and Prakash et al., 2005; Table 1). The reason for this diversity Trf5 proteins of Saccharomyces cerevisiae exhibit poly(A) RNA seems to be that polymerases have become specialized by polymerase activity but no DNA polymerase activity. CPD=cyclo- modifying the characteristics of the polymerization reaction butane pyrimidine dimmer. or by acquiring additional biochemical functions to tailor individual enzymes to particular tasks. In contrast to the different subunits. However, in each case one of the sub- single polymerase responsible for bacterial chromosome units is easily identifiable as containing the polymerase replication, three polymerases (a, d and e) are needed for catalytic site and this allows classification of polymerases eukaryotic chromosome replication (Table 1), and even more on the basis of amino acid sequence similarities. Eukaryotic polymerase diversity is needed for DNA repair (Table 1). DNA polymerases fall into five families, designated A, Polymerases involved in chromosome replication may also which also includes the archetypal Escherichia coli DNA participate in repair pathways, such as nucleotide excision polymerase I; B, which includes eukaryotic polymerases repair. Other types of repair synthesis involve specialized involved in bulk chromosome replication and RT, X and polymerases, some of which are proficient at replicating Y, which include enzymes involved in DNA repair or through a lesion, and this translesion synthesis (TLS) may be specialized types of replication (Table 1). On the basis of mutagenic. See also: Bacterial DNA Polymerase I sequence comparisons, polymerases in different families Some eukaryotic DNA polymerases consist of a single may have limited similarity, but polymerases in families A polypeptide chain, whereas others, such as those involved and B appear to have an identical mechanism for the in chromosome replication, are composed of several polymerization reaction (see the following section). 2 ENCYCLOPEDIA OF LIFE SCIENCES & 2009, John Wiley & Sons, Ltd. www.els.net Eukaryotic DNA Polymerases Polymerases Involved in Chromosome single-stranded incisions on each side of the damaged/ mismatched region. This allows removal of a short oligo- Replication nucleotide (24–32 nucleotides) and the resulting single- stranded region can then be copied by pol . Ligation of the Chromosomes are replicated during the S phase of the cell d nick completes the repair. cycle by three multisubunit polymerases – DNA pols a, d and e – which are found in all eukaryotes. The process of Pol « DNA replication involves initiation, which involves pol a, when DNA synthesis is activated at multiple replication Pol e consists of four subunits and probably functions at the origins in the chromosomes, and elongation, which involves leading strand (Kunkel and Burgers, 2008). Like pol d, pol e extension by pols d and e of the short DNA chains generated has a 3’–5’ exonuclease activity that provides a proofreading during initiation by pol a. A summary of the properties of function. Pol e is a processive enzyme by itself and it is not these polymerases is provided in the following sections; for clear whether it needs to interact with PCNA for function detailed
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