What proteins separate the 2 strands of DNA for DNA polymerase so as to give it a single stranded template?

Abstract

DNA polymerases are the only enzymes capable of duplicating the genetic information stored in the nucleic acid DNA, generating a faithful copy. As a consequence, they are essential for replicating the entire genome of any living organism before cell division, as well as for maintaining the integrity of the genetic information during the entire life of each cell. All organisms, either unicellular or pluricellular, that use DNA as their genetic information require one or more DNA polymerases for their survival. Most DNA viruses encode their own DNA polymerases, while a few predate those of the infected cells for replicating their genomes. Hence, DNA polymerases are essential for all kingdoms of life.

Summary

DNA polymerases cannot initiate synthesis of DNA chains in the absence of a priming device. Although DNA polymerases can readily extend short DNA primers that are randomly annealed to the template strand in vitro, specific priming is observed in vivo. In vivo primers may be the following: RNA synthesized by RNA polymerases, by primases or the RNA is pre-formed (tRNA); DNA produced by combination of an initial RNA primer and then extension by a DNA polymerase as observed for the dual action of primase and DNA polymerase α in eukaryotes; DNA primers produced by specific nicking of one strand of DNA or by recombination; and terminal proteins.

5 Conclusions and perspectives

DNA pol is a key enzyme in the replicative cycle of herpesviruses, and constitutes an attractive target for the development of potent inhibitors. Most antiviral agents that are currently approved for the prevention and treatment of herpesvirus infections ultimately target the viral DNA pol of HSV-1, HSV-2, VZV and HCMV. Until now, no antiviral agents have been approved for the treatment of infections caused by HHV-6A, HHV-6B, HHV-7, EBV and HHV-8. Prolonged antiviral therapy may lead to the emergence of drug-resistant isolates that can retain pathogenicity and virulence. Mutations conferring drug resistance arise in genes encoding the viral enzymes that phosphorylate drugs and the DNA pol itself, with potential cross-resistance between antivirals. There is thus an urgent need to develop novel potent DNA pol inhibitors that demonstrate activity against drug-resistant isolates with an adequate safety profile. Flexible nucleoside analogs of ACV based on acyclic sugar synthesis were reported to overcome drug resistance and renewed interest in the development of nucleoside analogs [180]. Oligopeptides corresponding to the carboxyl-terminal part of UL30 or UL54 protein were shown to disrupt complex formation between the DNA pol and UL42 or UL44 processivity factor resulting in an inhibition of viral replication [181,182]. This suggests that blocking subunit interaction might be a potential strategy to develop DNA pol inhibitors with a novel mechanism of action. A better understanding of the impact of drug resistance mutations on the structure and biochemical activity of herpesvirus DNA pols may lead to a more rational structure-based molecular design of novel potent inhibitors. In a novel approach, a chimeric enzyme was engineered by mutating the active site of the DNA pol of RB69 bacteriophage to include nonconserved elements from helices N and P of herpesvirus enzymes [64,170]. In contrast to RB69 DNA pol, which is resistant to ACV-TP and FOS, the chimeric enzyme is sensitive to both drugs. The chimeric protein is readily expressed in Escherichia coli and used as a surrogate enzyme for herpesvirus orthologs to develop and evaluate novel potent DNA pol inhibitors [21]. More recently, new medicinal chemistry strategies such as virtual screening of compound databases by molecular docking to target proteins [183], structure–activity relationship [184], machine learning analysis [185] as well as fragment-based drug discovery approaches to generate optimized hits by using fluorescence-based binding assays [186] and X-ray crystallography [187] have been developed to identify novel compounds with potent activity against herpesviruses.

Other Thermostable DNA Polymerases

Another DNA polymerase isolated from Theimus aquaticus has been described (Chien et al., 1976; Kaledin et al., 1980). This enzyme has an approximate molecular weight of 62,000–68,000, a specific activity between 500 and 5200 U/mg, a temperature optimum of 70–80 ° C, and a pH optimum in the range of 7.8 to 8.3 (see Table 2). Optimal activity is obtained with 60–200 mM KCl and 10 mM Mg2 +. Based on the physical and biochemical characterization of the polymerases purified by Chien et al. (1976) and Kaledin et al. (1980), it is unclear whether these proteins are the products of a gene (or genes) distinct from that which encodes the DNA polymerase described by Lawyer et al. (1989, 1993), or are partially purified proteolytic degradation fragments of the same translation product.

Table 2. Properties of Other Known Thermophilic DNA Polymerases

Similarly, additional DNA polymerases have also been isolated from Thermus thermophilus (Tth) HB-8 (Rüttimann et al., 1985; Carballeira et al., 1990; see also Table 2). Rüttimann et al. (1985) characterized three DNA polymerase isoenzymes with molecular masses in the range of 110,000–120,000. These enzymes all lacked exonuclease activity. The three enzymes differ in their heat stability, thermal activity profile, and their ability to use manganese as a cofactor. A fifth Tth DNA polymerase reported by Carballeira et al. (1990) differs in molecular weight from the four described above. This 67,000-dalton protein lacks a 5′-to 3′-exonuclease and has a specific activity of 4000 U/mg.

The DNA polymerases from a number of other thermophilic eubacteria have also been isolated and partially characterized (Table 2). These include Bacillus stearothermophilus (Bst) (Stenesh and Roe, 1972; Kaboev et al., 1981), Bacillus caldotenax (Bca) (Uemori et al., 1993b), Thermus ruber (Tru) (Kaledin et al., 1982), Thermus flavus (Tfl) (Kaledin et al., 1981), and Thermotoga sp. (Tsp) strain FJSS3-B.1 (Simpson et al., 1990). In addition, other thermophilic archaeal DNA polymerases have been identified. A DNA polymerase has been purified and characterized from the thermoacidophilic arch-aeon Sulfolobus acidocaldarius (Sac) (Klimczak et al., 1985; Elie et al., 1988; Salhi et al., 1989). Similarly, DNA polymerases from Sulfolobus solfataricus (Sso) (Relia et al., 1990) and Thermoplasma acidophilum (Tac) (Hamal et al., 1990) have been isolated. The purification and characterization of a DNA polymerase from a methanogenic archaeon Methanobacterium thermoautotrophicum (Mth) (Klimczak et al., 1986) has also been reported.

Tables 1 and 2 show that considerable variation exists among those thermostable DNA polymerases that have been characterized in the literature. A distinct feature of these DNA polymerases is that they all appear to be monomeric. It is not known whether a multimeric DNA polymerase similar to E. coi DNA polymerase III exists in thermophilic bacteria. It is also not known whether these polymerases are involved in replication or repair. Two distinct DNA polymerase genes have been identified from Sso (Pisani et al., 1992; Prangishvili and Klenk, 1993), indicating that, like mesophilic organisms, multiple DNA polymerases are inherent to at least some thermophiles. A substantial difference among polymerases is the presence or absence of associated exonuclease activities. Only Taq DNA polymerase (Longley et al., 1990; Holland et al., 1991; Lawyer et al., 1993), Tth DNA polymerase (as isolated by Stoffel and Gelfand; Lyamichev et al., 1993; Aver et al., 1995), Tma DNA polymerase (Lawyer and Gelfand, 1992), Bca DNA polymerase (Uemori et al., 1993b), and Mth DNA polymerase (Klimczak et al., 1986) have been shown to contain an inherent 5′ to 3′–exonuclease activity, whereas only Tac DNA polymerase (Hamal et al., 1990), Tma DNA polymerase (Lawyer and Gelfand, 1992), Bca DNA polymerase (Uemori et al., 1993b), Mth DNA polymerase (Klimczak et al., 1986), Tli DNA polymerase (Kong et al., 1993), and Pfu DNA polymerase (Lundgren et al., 1991) have been shown to contain an inherent 3′-to 5′-exonuclease activity. Of the characterized thermostable DNA polymerases, only Mth DNA polymerase, Tma DNA polymerase, and Bca DNA polymerase resemble E. coli DNA polymerase I, in that they contain both a 5′- to 3′-exonuclease and a 3′- to 5′-exonuclease. The lack of any 5′- to 3′-exonucleolytic activity associated with many of the thermostable DNA polymerases may reflect a real difference among the enzymes (e.g., Family A- vs Family B-type polymerases), differences in the sensitivity of the assay procedures (see structure-dependent enhancement of activity, described later), or alternatively, may be the consequence of proteolytic degradation resulting in an amino terminal truncation of the protein. The absence of an inherent 3′- to 5′-exonuclease activity in a number of the polymerases may again reflect a real difference between species or type of polymerase characterized; alternatively a 3′- to 5′-exonuclease may exist as a separate subunit or auxiliary protein.

66.3.5 Polymerase Proofreading

DNA polymerases, which are multisubunit enzymes including Pol α, Pol δ, and Pol ε, are critical for the accurate replication of cellular DNA.277 While Pol α initiates DNA synthesis, Pol δ and Pol ε perform the majority of the DNA replication with Pol δ synthesizing the lagging strand and Pol ε synthesizing the leading strand. Given the importance of accurate DNA replication, the proper function of these enzymes is critical to maintain DNA stability. Recently, mutations in the DNA polymerases have been discovered as a mechanism of inducing genetic instability, especially mutations that involve the catalytic and proofreading subunits of Pol δ and Pol ε, POLD1 and POLE, respectively.278 Germline mutation in either POLE or POLD1 can lead to colonic polyposis and an increased risk of both colorectal cancer as well as endometrial cancer. This cancer predisposition is inherited in an autosomal dominant fashion, and the polyposis associated with these mutations has recently been referred to as polymerase-proofreading associated polyposis (PPAP).279 Tumors resulting from DNA polymerase proofreading dysfunction are typically hypermutated; however, they remain MSS.

In addition to germline mutations in POLD1 and POLE, somatic mutations in POLE are also important in sporadic colorectal cancer. In fact, it was recently shown that 3% of colorectal cancers had mutations in the exonuclease domain of POLE.92 Given the recent discovery of the role of the DNA polymerases in colorectal carcinogenesis, at this time it remains unknown whether defects in these polymerases are important for the pathogenesis of other types of gastrointestinal cancer.

Priming DNA Synthesis

DNA polymerase cannot initiate new strands of nucleic acid synthesis because it can only add a nucleotide onto a pre-existing 3′-OH. Therefore, an 11 to 12 base-pair length of RNA (an RNA primer) is made at the beginning of each new strand of DNA. Since the leading strand is synthesized as a single piece, there is only one RNA primer at the origin. On the lagging strand, each Okazaki fragment begins with a single RNA primer. DNA polymerase then makes DNA starting from each RNA primer. At the origin, a protein called PriA displaces the SSB proteins so a special RNA polymerase, called primase (DnaG), can enter and synthesize short RNA primers using ribonucleotides. Two molecules of DNA polymerase III bind to the primers on the leading and lagging strands and synthesize new DNA from the 3′ hydroxyls (Fig. 4.5).

Primase, a special RNA polymerase, works with PriA to displace the SSB proteins and synthesize a short RNA primer at the origin. DNA polymerase then starts synthesis of the new DNA strand using the 3′-OH of the RNA primer. This synthesis occurs at multiple locations on the lagging strand.

1 Introduction

DNA polymerases play a pivotal role in DNA replication and DNA repair by catalyzing the stepwise addition of deoxyribonucleotides to a growing DNA chain. The complexity of the cellular environment and resultant requirements on adaptability and specificity led to a profound differentiation of DNA polymerases (Baker & Kornberg, 1992; Hübscher, Spadari, Villani, & Maga, 2010a, 2010b), exemplified by a staggering number of 18 different DNA polymerases functioning in human cells (García-Gómez, Reyes, Martínez-Jiménez, et al., 2013). Here, we discuss studies of the prokaryotic DNA polymerase I (hereafter Pol I), which we have been using as a model system to investigate the influence of the polymerase’ conformational landscape on the fidelity of DNA synthesis by means of single-molecule fluorescence techniques (Fig. 1).

The Escherichia coli Pol I was the first DNA polymerase discovered (Lehman, Bessman, Simms, & Kornberg, 1958) and features three enzymatic activities: the 5′-to-3′ polymerase activity, which is responsible for DNA synthesis; the 3′-to-5′ exonuclease activity, which allows the removal of wrongly incorporated bases; and the 5′-to-3′ exonuclease activity, which allows the polymerase to remove flaps of downstream DNA created as a result of strand displacement by Pol I. Much of our structural and mechanistic knowledge on Pol I comes from studies of a cleavage product of full-length Pol I, known as Klenow fragment (KF), which lacks the 5′-to-3′ exonuclease activity (Joyce & Grindley, 1983; Joyce, Potapova, DeLucia, et al., 2008). As expected for an enzyme involved in gap filling and strand displacement of RNA primers in DNA replication, Pol I features a remarkably high fidelity: insertion errors are extremely rare and occur only at a rate of 1 in ~ 106 additions (Bebenek, Joyce, Fitzgerald, & Kunkel, 1990; Eckert & Kunkel, 1991). The overall structure of Pol I (KF) resembles the form of a human right hand and consists of four subdomains: the 3′-to-5′ exonuclease, the thumb, the palm, and the fingers subdomain, with the latter playing an important role in nucleotide selection and incorporation (Fig. 2A).

Superimposing the high-resolution cocrystal structures of a KF homologue binary structure (DNA polymerase + DNA) with the ternary structure (DNA polymerase + DNA + complementary nucleotide) revealed a large conformational change of the fingers subdomain upon nucleotide addition (Fig. 2A) (Johnson et al., 2003). This inferred conformational change has been the starting point of our work, which has been largely driven by the question how exactly conformational changes contribute to polymerase fidelity. In the following, we will describe the individual steps necessary to address the research question that did not only require the optimization of existing biochemical protocols for labeling DNA polymerases with fluorophores but also the development of new tools for single-molecule spectroscopy. We will also link our work to recent advances in the field of single-molecule fluorescence detection, providing the interested reader with a brief overview over alternative approaches to study molecular machines and DNA polymerases at the single-molecule level.

Multiple Enzymes and Factors Are Involved in the DNA Replication Process

The replication process is exquisitely regulated to ensure that the DNA in each cell is duplicated only once per cell cycle, during the S phase. Cyclins and cyclin-dependent kinases are involved in this regulation. Although the basic mechanism is similar, replication is more complex in eukaryotes than in prokaryotes.

The initial step of replication is the separation of both DNA strands. This is necessary to copy the parental DNA, which functions as a guide for the assembly of the new complementary strand.

Replication begins at a defined point of origin, or initiation site, where specific base sequences serve as recognition signals for the enzymes and factors that initiate replication. Many initiation sites contain sequences rich in A-T pairs, which are easier to separate than G-C pairs. The first step is the formation of the origin recognition complex (ORC), composed of six subunits. Then, the mini-chromosome maintenance complex (MCM), also formed by six subunits, is added.

Prokaryotic chromosomes, mitochondrial DNA, and circular viral DNA have a single site of origin. In contrast, linear eukaryotic DNA molecules in chromosomes begin to replicate at multiple sites. In humans there are between 30,000–50,000 initiation sites. Separation of the DNA strands is initiated simultaneously in all chromosomes and at many different points along the molecule. As a result, the double helices form “bubbles” at the separation zones. These bubbles or replication units are called replicons. In the nucleus of every human cell about 50,000 “replication bubbles” can be formed. This simultaneous unwinding of DNA in many different sites is completed faster than if performed progressively from one end to another of the very long double helix of each chromosome.

The site where both strands are separated is called the replication fork. In each separation area, two forks are formed, which progress in opposite directions, away from the point of origin (Fig. 21.2).

Helicase. Also called the unwinding enzyme, helicase catalyzes the separation of the DNA strands. The energy required for the process is provided by the hydrolysis of ATP.

Topoisomerases. In bacterial circular DNA, the separation of the DNA strands at a single site produces supercoiling in other regions of the double helix. In short linear DNA molecules, the separation does not create tensions; these are easily relieved by rotation of the free ends. In contrast, due to the enormous length and multiple interactions in the nucleosomes, the linear DNA molecules of eukaryotes do not rotate freely, and torsions frequently occur downstream of the replication fork. In both bacteria and eukaryotes, supercoiling is resolved by periodical cuts in the chain, in areas where torsions occur. These cuts are catalyzed by topoisomerases, of which two types (I and II) are known. Isomerase type I cuts one strand of the double helix and relieves tension within the molecule by rotation of the noncut end on the other strand. No energy is required as the passage of the supercoiled to the relaxed state of DNA has a negative ΔG. The type II enzyme, also called gyrase, cuts both DNA strands. It is dependent on the hydrolysis of ATP. Both enzymes also have the capacity to bind back the cut ends of the chains and to reestablish the double helix once the relaxed state is attained.

Bacterial gyrase is inhibited by antibiotics, such as nalidixic acid and other substances. Eukaryotic topoisomerase II is unaffected by nalidixic acid, so, this compound is useful for the treatment of human infections caused by bacteria that are susceptible to this type of antibiotic.

Specific proteins maintain the DNA strands separated. As the double helix strands are separated, they bind to single-strand DNA binding proteins. These proteins in bacteria are designated with the acronym SSB. They stabilize and prevent chain reannealing. The binding of the SSB does not disturb the “copying” process of a complementary strand synthesis. In eukaryotes, this function is performed by replication protein A (RPA).

Helicase and binding proteins move along the double helix leaving behind two separate chains, ready to serve as a template for the synthesis of new complementary strands. Synthesis is initiated simultaneously at all sites of unwinding, before the original double helix is fully separated (Fig. 21.2).

DNA polymerases. Formation of the new chain is performed by assembly of deoxyribonucleotides catalyzed by DNA polymerases. In bacteria, three of these enzymes have been isolated, each designated with Roman numerals (I, II, III). In eukaryotes, a higher number of polymerases have been identified. They are named by Greek letters; the most important ones include the α, β, γ, δ, and ɛ polymerases. All DNA polymerases are molecular complexes formed by association of different subunits. They differ in their properties and functions; however, they all share the following characteristics:

Their substrates are the four deoxyribonucleoside triphosphates dATP, dGTP, dCTP, and dTTP. These molecules not only provide the “raw material” for the synthesis, but also the energy required for their assembly.

They need a free strand of DNA that serves as a guide or template. They only work on a single DNA strand to insert, one by one, nucleotides that are complementary to those in the template strand.

They are unable to bind free nucleotides and start a new strand. They can only extend a preexisting strand (the initiator or primer strand) correctly paired by hydrogen bonds with the template DNA bases.

All DNA polymerases catalyze the binding of deoxyribonucleotides to form chains in the 5′ to 3′ direction. This involves establishing diester connections from the 3′ carbon hydroxyl in the terminal nucleotide deoxyribose of the initiator-chain to the 5′ carbon phosphate of the entering nucleotide. However, the helices are antiparallel, so, there are polymerases that act on the chain that serves as a template in the 3′→5′ direction, which is opposite to the synthesis of the new strand.

Replisome. All proteins involved in DNA replication form a multienzyme complex that functions as a “replication machine”; this is called the replisome.

i. Primary functions of DNA polymerases.

DNA polymerases are a group of polymerases that catalyze the synthesis of polydeoxyribonucleotides from mono-deoxyribonucleoside triphosphates (dNTPs), performing the most fundamental functions in vivo of DNA replication, repair, and, in some cases, cell differentiation. In fact, different types of DNA polymerases have been found in a single organism, for example, three (DNA Pol I, II, and III) in E. coli or five (DNA Pol α, β, γ, δ, and ɛ) in higher eukaryotes, which are believed to perform a specialized in vivo function(s). With somewhat different complexities from in vivo functions, DNA polymerases can, when given suitable conditions, also perform DNA synthesis in vitro. They require, in addition to dNTPs, an initiating oligonucleotide (or polynucleotide), called a primer, carrying a 3′-end hydroxyl group that can be used as the starting point of chain growth (1, 2). DNA polymerases cannot initiate synthesis de novo from mononucleotides.

A primer can be a short or long piece of DNA or RNA which carries a free 3′-OH group. Primers provide a double-stranded structure to the DNA polymerase by annealing to a complementary region of the DNA or RNA strand called a template. The DNA polymerase moves along the DNA (or RNA) template, extending the primer in the 5′ → 3′ direction according to the Watson–Crick base pairing rule, i.e., A pairs with T (or U) and C pairs with G (see Section II, Chapter 1). The polarity of the newly synthesized chain is opposite (or antiparallel) to that of the template. Incorporation of a noncomplementary nucleotide is considered an “error.” The error frequency (or fidelity) is an important characteristic of a polymerase (see below).

In addition to the major 5′ → 3′-polymerase activity, a DNA polymerase may exhibit several other activities, such as 5′-nuclease, 3′ → 5′-exonuclease, and/or RNase H activities, which are necessary for proper in vivo functions.

The initiation of cellular DNA replication takes place at a single site (e.g., oriC of E. coli) or multiple specific sites (in higher eukaryotes) of DNA called origins of replication (ori). The temporal site of dsDNA where the replication occurs is called a “replication fork.” Because of DNA strand polarity, the bidirectional replication results in two distinct products, “leading” and “lagging” strands, according to the moving direction of the replication fork. The leading strand is synthesized as a single continuous chain, whereas the lagging strand is initially synthesized as small oligonucleotides, called Okazaki fragments, which are then ligated to form a continuous chain. Small RNAs play an important role as natural primers in the synthesis of both the leading strand and, in particular, the lagging strand.

Replication of duplex, RF DNA of single-stranded bacteriophages and of plasmids is initiated at a site of single-strand scission, called a nick, which is site specifically introduced by an endonuclease. The extension of a primer (or positive) strand on the template (minus) strand proceeds by a rolling circle mechanism in which a “daughter” strand longer than one full length of the template circle is produced and then processed.

3.9 DNA Polymerase λ

DNA polymerase λ mediates DNA repair processes by catalyzing the polymerization of deoxyribonucleotides alongside a DNA strand. Curcumin has been reported as a potent inhibitor of polymerase λ in laboratory studies. Curcumin inhibited polymerase λ and suppressed the growth of human NUGC-3 cancer cells by mediating cell cycle arrest at the G2/M phase [68]. The same group of researchers also synthesized MAC, which bounds noncompetitively and more potently inhibited pol λ inhibitor than curcumin [69]. Computational molecular docking analyses revealed that MAC bound selectively to the N-terminal domain of pol λ, which consisted of a β-sheet (Thr51 of sheet-1), an α-helix (residues 57–69), and the two loops (residues 51–56 and 70–75) [70]. This study also demonstrated that MAC did not bind to the C-terminal region. Further studies are need to determine the binding of curcumin and curcumin derivatives with DNA polymerase λ.