2) DNA polymerases 2.1) General
The survival and development of each organism relies on the equal distribution of its genome during cell division. Errors in this process can lead to severe developmental defects, cancer, or even death.15‐17 DNA polymerases are key enzymes to pass the exact genomic information down generations. Over 50 years ago, Kornberg et al. discovered in E. coli the first enzyme (DNA polymerase I or Kornberg‐Polymerase), that catalyses the accurate replication of DNA.18,19 Since this and other pioneering discoveries, it was assumed for a long period of time, that only six “classical” DNA polymerases (pol α, β, γ, δ, ε, and terminal deoxynucleotidyl transferase (TdT)) are responsible for DNA replication and repair in all mammalian cells.15,17,20,21
For that reasons, the discovery of several “novel” specialized DNA polymerases was a real sensation in the last decades.15,20,22 So far, at least 15 different human DNA polymerases are known.15,17,20,22
All enzymes share a common 3D‐structure, that is reminiscent of a right hand, and can be subdivided into a finger‐, thumb‐ and a highly conserved palm domain (see also Figure 4).17,20,23,24 With regard to their sequence homology and structural similarity, the 15 enzymes have been subdivided into six DNA polymerase families A, B, C, D, X, and Y (Table 1).20,25,26
The basic functions of the six “classical” DNA polymerases have been elucidated from catalytic properties, and observation of cell physiology. Pol α catalyses the initiation of chromosomal DNA replication at origins of replication and at Okazaki fragments on the lagging‐strand,27,28 pol β is involved in base excision repair (BER),29‐31 pol γ synthesizes mitochondrial DNA,32 pol δ has a role in lagging‐strand synthesis,33,34 pol ε participates in the synthesis of the leading‐strand of chromosomal DNA,35 and TdT facilitates antigen receptor diversity36.15,20
In the course of these and other cellular processes or by environmental conditions, DNA mutations and damages occur.16 To maintain the genetic integrity of the genome, an elaborate set of sophisticated repair mechanisms have evolved. The set includes, amongst others, the “novel” specialized DNA polymerases (pol η, θ, κ, λ, μ, ν, ι, ζ, and REV1).15‐17,20,37
Features of some of these enzymes are known (Table 1), but to understand in depth the task of a particular enzyme stills await clarification in the majority of the cases. For that reason, there is a great demand for appropriate methods and reagents (e.g. small‐molecule probes) to dissect the cellular functions of DNA polymerases.
Table 1. Human DNA polymerasesa
DNA polymerase Gene Protein size (kDa) Family Main Function
pol α (alpha) POLA1 166 B DNA replication priming
pol β (beta)b POLB 38 X DNA repair
pol γ (gamma) POLG1 140 A Mitochondrial DNA replication and repair pol δ (delta) POLD1 124 B DNA replication (lagging‐strand)
pol ε (epsilon) POLE 262 B DNA replication (leading‐strand) TdT DNTT 58 X DNA repair, V(D)J recombination
pol η (eta) POLH 78 Y Bypass synthesis (inserter)
pol ι (iota) POLI 80 Y Bypass synthesis (inserter)
pol κ (kappa) POLK 99 Y Bypass synthesis (inserter/extender) pol λ (lambda)b POLL 63 X DNA repair, V(D)J recombination
pol μ (mu) POLM 55 X DNA repair, V(D)J recombination
pol θ (theta) POLQ 290 A DNA repair
pol ζ (zeta) POLZ 353 B Bypass synthesis (extender)
REV 1 REV1 138 Y Bypass synthesis (inserter)
pol ν (nu) POLN 100 A DNA repair
a table was adapted from literature 15,17,20.
b see also chapter 2.3)
2.2) DNA polymerases and the polymerisation reaction
All previously discovered DNA polymerases share a common, over several steps well‐
coordinated reaction mechanism (Figure 2) for the synthesis of the helical DNA polymer (composed of a sugar phosphate backbone, to which the four heterocyclic bases adenine (A), guanine (G), thymine (T), and cytosine (C) are attached38) (Figure 3).38‐42
E:DNAn
E
E:DNAn:dNTP
E*:DNAn:dNTP
E*:DNAn+1:PPi
Figure 2. Mechanism of DNA polymerase catalyzed nucleotide incorporation. After binding of a DNA primer template complex (DNAn), the DNA polymerase (E) binds an incoming dNTP that is afterwards tightly bound and arranged for the chemical step by a conformational change of the enzyme (E*). After bond formation and an other conformational change, pyrophosphate (PPi) is released to start another cycle of catalysis. Figure was adapted from literature 41‐43.
Figure 3. Enzymatic DNA polymerization. (A) Schematic representation of template directed DNA synthesis catalyzed by a DNA polymerase. (B) Schematic representation of the corresponding trigonal bipyramidal transition state in the active site of a DNA polymerase (template not shown). Figure was adapted from literature 24,44.
DNA replication proceeds semiconservative. In doing so, DNA polymerases use one DNA parent strand as a template for synthesis of the exact complementary replica.18,20,45 During the synthesis, the single‐stranded template dictates to the enzyme according to
Watson‐Crick38, in which sequence the four native 2´‐deoxynucleoside‐5`‐triphosphates (dNTPs) have to be connected to the 3`‐OH end of the hybridized primer. Thereby, the primer is always extended in the 5` to 3` direction (Figure 3A).20,42
From a chemical point of view, the addition of a dNTP to the primer is performed according to the mechanism of nucleophilic substitution (SN2). In the active site of the enzyme, the trigonal bipyramidal transition state is stabilized by two metal ions, e.g. magnesium (II) ions.
For that reason, the reaction mechanism is also called the “two metal ion mechanism”
(Figure 3B).24 The SN2 reaction is accompanied by pyrophosphate release. Its subsequent hydrolysis favors DNA synthesis and prevents the reverse reaction. According to the latest findings, the rate limiting step of the reaction is a local reorganization step in the active site of the enzyme.46 Nevertheless, the velocity of the polymerisation (kpol) of correct paired nucleotides achieves 1000 dNTP s‐1, and the catalytic efficiency (Kd kpol‐1) is in the region of diffusion control (~107 M‐1 s‐1).41,42 Due to this facts, DNA polymerases belong to most powerful enzymes.47
2.3) DNA polymerase λ and β
Most of the work presented herein covers the “classical” pol β31,32,48 and the recently discovered “novel” pol λ49. Both nonreplicative human enzymes are members of the DNA polymerase X‐family (Table 1).15,17,20,22,37
The exonuclease‐deficient pol λ (64 kDa) contains all the structural features required for DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, which are conserved in pol β (39 kDa) ‐ the smallest known human DNA polymerase (Table 1). On this account, the primary sequence and the 3D‐
structure of the catalytic core of both DNA polymerase are highly homologous (Figure 4).49,50 Because of its ability to remove the 5`‐deoxyribose phosphate (dRP) generated after incision by an abasic (AP) endonuclease (dRP‐lyase activity) and its DNA synthesis specificity for short gaps, pol β is the prime DNA polymerase participating in BER (Figure 5).15,17,20,30,37,51
In addition, pol β is able to associate with other downstream enzymes of the BER pathway like DNA ligase I, AP endonuclease, and XRCC1‐DNA ligase III.30,37 Extensive studies show that pol β bypasses several DNA lesions via translesion synthesis (TLS), for example, AP sites52 and cisplatin adducts53.
Figure 4. Family X DNA polymerase λ and β. (A) Schematic representation of pol β (red) and pol λ (green). Pol λ consists of a nuclear localization signal (NLS), a BRCA1‐C terminal (BRCT) domain (residues 36‐132), a proline‐
rich region (residues 133‐243), and a pol β‐like catalytic core region (residues 244‐575), with a helix‐hairpin‐
helix (HhH) and a DNA polymerase X motif.49 (B) Superimposition of the pol β‐like catalytic core region (residues 244‐575) of pol λ (green) and pol β (red). PDB IDs 2PFN and 2FMP (shown without DNA).
Pol λ, the other DNA polymerase of interest, is unique in possessing all the enzymatic activities which are individually present in the other X‐family members.20 Pol λ is capable of synthesizing DNA de novo as well as template‐dependent, and displays dRP‐lyase and TdT activity.54‐60 It is implicated that pol λ is involved in gap filling during nonhomologous end joining36,61,62, TLS52,63,64, and BER54,65,66.
Moreover, studies with chicken DT40 cells67, as well as mammalian fibroblasts68, showed that pol λ has a backup role for pol β in BER. Experiments with reactive oxygen species (ROS) indicate that pol λ protects cells from oxidative damage66,69, and there is also evidence that pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells.69
Figure 5. Schematic representation of BER imbalance by targeting pol β. BER is a highly coordinated, multistep pathway, that removes a damaged DNA base and replaces it with the correct base. The genotoxic TMZ induce the formation of a base damage (e.g. 3‐methyladenine (3meA)), which is excised by DNA glycosylase (AAG) to produce an apurinic site (AP). Afterwards, an AP endonuclease (APE) incises the DNA backbone (5´ to the AP site) and generates a single‐strand break. Then, pol β removes the 5´‐dRP moiety through its intrinsic lyase activity and fills in the resulting gap. In the final step, the nick is sealed by DNA ligase (LIG) to finish base excision repair (BER). If BER is inhibited, or downstream steps of BER are limiting, then toxic intermediates accumulate and can lead to cell death. In this way, the dosage of the DNA‐damaging agent can be reduced.
Figure was adapted from literature 16,70.
It is well known that aberrant levels of specialized DNA polymerases might cause genomic instability.15,20,71 A recent investigation of the expression patterns of specialized DNA polymerases in 68 different tumor samples revealed that in more than 45% of these tumors at least one specialized DNA polymerase was two‐fold‐enhanced expressed. Of particular interest was the fact that over 30% of all samples had either pol λ or β overexpressed.72 Consequently, the regulation of both DNA polymerases could be crucial in cancer treatment, since many chemotherapeutic regimes in use depend at least in part on the artificial induction of DNA damage. If effective, their utility is typically limited by the severity of side effects caused by the nonselective targeting of cancerous and healthy tissue in addition to the potential to induce mutagenic events that can actually accelerate disease
development.41,70,73 The clinical efficacy of anticancer drugs like cisplatin and monofunctional alkylating agents (e.g. temozolomid (TMZ)) is often reduced by cellular DNA repair mechanisms.15‐17,20,37,51,70
Consequently, both enzymes specialized for DNA repair are discussed as promising future drug targets, to reduce the dosage of DNA‐damaging agents while improving their activity via targeting of their respective repair pathways (Figure 5).15,17,20,37,51,70,74
To date, several inhibitors of DNA polymerase λ and β were developed and investigated (see also chapter 4.4.4).37,51,74‐79
However, one remaining challenge is still to find novel potent small molecule inhibitors that selectively inhibit one of these enzymes. In addition, a discriminating inhibitor could facilitate the targeting of one of these DNA polymerases over the other, to probe the enzymes’ respective cellular functions.
2.4) Herpes virus DNA polymerase
Viral infections are the leading cause of many critical illnesses. Without exception, all viruses are obligate, intracellular molecular parasites that replicate only inside the cell of a living organism.80,81 For the reproduction of the viral genome, viruses often encode an own DNA polymerase.20 Due to the importance of these enzymes for the amplification of the genetic code, viral DNA polymerases are established targets for current chemotherapies.41,81 These facts are also relevant for the family of the Herpes viruses (see also chapter 5.1), which reproduce their genetic code with their own DNA‐dependent DNA polymerase in the nucleus of a host cell. Among the nine representatives of the morphologically very similar Herpesviridae, herpes simplex virus‐1 (HSV‐1) is the most researched member.20 For that reason, the HSV‐1 DNA polymerase serves herein as model enzyme, which is illustrated briefly. After the HSV‐1 infection of mammalian cells, Keir et al. discovered in 1966 for the first time DNA polymerase activities that deviated from the features of host enzymes.82 Later, Weissbach et al. purified and characterized a viral DNA polymerase with a high molecular weight (180 kDa) from HSV‐1 infected human cells.83 It was found, that the enzyme favoured
‐ very likely due to the composition of the HSV genome (67% GC content)20 ‐ the synthesis of
GC rich DNA.83 Today, the 3D‐structur of the HSV‐1 DNA polymerase is known. The enzyme is a heterodimer, composed of a catalytic subunit (UL30), whose structure is similar to B‐family DNA polymerase structures, and a processivity factor (UL42) that increases the fidelity.84‐86 The catalytic subunit consists of six domains. The N‐terminal part contains a pre‐N‐terminal,
an N‐terminal, and an exonuclease domain, that ensures a high fidelity of DNA replication as well as an RNase H activity.20,87 The C‐terminal part adopts the usual right hand folding in palm, fingers and thump domains.85 Based on sequence alignments, the replicative HSV DNA polymerase (UL30) belongs to the B‐family of DNA polymerases (see also chapter 2.1).20