Eukaryotic DNA Polymerases
The DNA polymerases of eukaryotes are in general less understood than the DNA polymerases of prokaryotes, but recently much has been learned about their functions and activities. DNA polymerases from yeast and mammalian cells (especially mouse and human) are among the best studied. Eukaryotic cells have at least four major nuclear DNA polymerases: α , β, δ, and ε . Polymerase γ is found in mitochondria, although it is encoded by a nuclear gene. Plant chloroplasts also contain their own DNA polymerase that appears to be similar to γ . The six polymerases mentioned so far are involved in DNA replication, DNA repair, or both. These "classical" pols can be characterized as accurate. Most of these have been known for some time; for example pol α was discovered in the late 1950's, and pols δ and ε in the 1980's. However, since 1999, at least 10 new cellular pols have been discovered. These pols are mostly innacurate. They are not involved in chromosomal replication, but in other essential processes such as inaccurate translesion repair and somatic hypermutation of immunoglobulin alleles. We will discuss these later, when we cover repair processes. Here we will focus on the classical, nuclear DNA polymerases, as well as some interesting viral pols. In addition, because of complexity of cellular genome and cellular DNA replication, viral model systems have proved invaluable. Among the most useful of these is simian virus 40 (SV40). SV40 is a small mammalian virus with a circular duplex genome of about 5 kbp. It does not encode a polymerase and thus uses host DNA polymerases to replicate its genome. Both cell-free systems (cell extracts plus viral DNA templates) and in vitro, reconstituted replication systems using viral DNA with purified cellular proteins have been studied. This has allowed the identification of DNA polymerases and accessory factors required for replicative DNA synthesis in host cells. Other important viral model systems include the E. coli phages T4 and RB69, and the mammalian viruses herpes simplex virus (HSV) and adenovirus. These viruses encode their own pols and accessory replication proteins.
Ann. and enzyme activity is present in cycling cells. ed. and the only one capable of selfprimed DNA synthesis on a previously unprimed ssDNA. bridge with DNA pol (protein subunit sizes from human cells)
.-F.) Cold Spring Harbor Laboratory Press. but is low or undetectable in differentiated. Pol α function is required for cell viability and nuclear DNA replication. Xenopus laevis (African clawed toad).. (1996) Cellular DNA polymerases. 71:133163. (2002) Eukaryotic DNA polymerases. G. (Reviews: Wang. Because of this.structural. • Pol α is an essential component of the reconstituted in vitro SV40 replication system.S. 28). protein. Biochem. Pol α polymerase and primase activities This unique enzyme has two distinct polymerase activities: a 5’→ 3’ DNA-dependent DNA polymerase. The RNA polymerase activity is a primase. U. protein-protein interactions 48 kDa -. • Depletion of Pol α by antibody precipitation blocks SV40 DNA replication in HeLa (human) cell extracts. T. In: “DNA Replication in Eukaryotic Cells” (M. S. It is the only enzyme known to have both DNA polymerase and primase activities. Pol α :primase is a heterotetramer: 180 kDa – DNA pol catalytic subunit 68 kDa –. the enzyme is often referred to as Pol α :primase. (Fig. and mouse.In addition to the human system and various viral models. Hubscher. important model organisms for cellular DNA synthesis include Saccharomyces cerevisiae (budding yeast) Schizosaccharomyces pombe (fission yeast). and a 5’→ 3’ DNA-dependent RNA polymerase. • Pol α transcript. DePamphilis. In yeast. quiescent cells (G0) in all systems examined.. for example: • ts Pol α mutants do not replicate DNA at restrictive (or non-permissive) temperature (cultured mouse cells and yeast). Rev.) DNA polymerase α A wealth of genetic and biochemical evidence indicates that this polymerase is required for chromosomal replication. Maga. and Spadari.primase catalytic subunit 55 kDa – primase subunit. mutants are blocked at S-phase in the cell cycle at non-permissive temperature.
The substrates for the DNA polymerase reaction are a template primer (iRNA is the primer) and dNTPs. or iDNA). with an average length of ~40 nt. genetic analysis indicates that the primase subunit is required for cell viability and nuclear DNA replication. Pol α does not have an intrinsic 3'→ 5' exonuclease activity and is not known to associate with one (no apparent editing activity). with the structure pppRNAn -p-DNAn .) Eukaryotic pols can generally use DNA or RNA primers. it is composed of 4 subunits: 125 kDa-. its DNA pol activity is not a very processive and it does not associate with a clamp. Why are RNA/DNA primers used in eukaryotes? (Recall that prokaryotes use primers that consist of RNA only.pol catalytic subunit 66 kDa-. The DNA polymerase activity is also not very processive. that are complementary to an ssDNA template.The primase activity is not very processive. The substrates for the primase reaction are ssDNA (unprimed) and rNTPs. DNA polymerase δ Pol δ is another multisubunit nuclear enzyme. protein-protein interactions
. the primary function of Pol α :primase is to make short RNA/DNA primers for replicative DNA synthesis. It incorporates rNMPs to synthesize short RNA primers. the Pol α polymerase activity is known to be essential for replicative DNA synthesis. These oligoribonucleotides are called initiator RNA (iRNA). It is probably not involved in significant primer elongation. This also applies to the primase activity.) Physiological role of Pol α :primase As indicated previously. In yeast. but rNTPs are preferred. The final product of both activities is an RNA/DNA primer. The primase will use dNTPs as substrate if rNTPs are not available. But eukaryotic clamp loaders generally prefer DNA primers. In Homo sapiens.structural. In vivo. synthesized by DnaG primase. (The DNA is sometimes called initiator DNA. Typically.interaction with processivity factor. (Eukaryotic polymerases in general lack this activity. Pol α adds about 30 dNMPs to the 3' end of the iRNA primer. multimerization (?) 50 kDa-. It also lacks a 5' → 3' exonuclease activity. Its inability to edit may not a liability because both the RNA and DNA portions of the primers are mostly removed and replaced during chromosomal replication (discussed later). Accordingly. about 8-12 nt long.
. J. Not surprisingly.12 kDa-. and it is PCNA that acts as a processivity
factor. see : Kelman. So unlike the situation in Pol III. coli Pol III holoenzyme and phage T4 gp45 in structure. The 66 kDa subunit is needed for interaction between Pol δ and PCNA. T. so Pol δ. J.. Hurwitz. Z. (1998) Processivity of DNA polymerases: two mechanisms.R. Kong.) Replication factor-C (RF-C) Like β and gp45. S. 30). PCNA functions as a sliding clamp to increase the processivity of Pol δ up to 50-fold. Gary. M.M. called proliferating cell nuclear antigen (PCNA). forming a pre-initiation complex. Pol δ requires an associated 30 kDa protein. whereas β is a homodimer. Functional PCNA is a homotrimer (120 kDa) and acts as the processivity factor. In the presence of PCNA. the structures of PCNA. Pol δ (via the 66 kDa subunit) then interacts with PCNA and the primer terminus to form an initiation complex. proteins of the RF-C complex show functional and structural homology to the γ complex of Pol III and the simpler gp44/62 complex of T4. The smaller subunits appear to be involved in holding the multisubunit structure together (via protein-protein interactions). The RF-C complex consists of five subunits which together load PCNA onto the template primer in an ATP-dependent reaction. T4..-P. and β are virtually superimposable. may also be a dimeric replicating machine. Cell 79: 1233-1243.. gp45. although there is little sequence homology (Fig. for full polymerase activity and processivity. Pol
is highly processive. It is similar to the β subunit of E. The reactions these complexes catalyze are very similar (Fig. Thus Pol δ is a typical replicase complex. 29).structural. and Kuriyan. Pol δ. like Pol III. for a review of Pol III. In fact. Structure 6: 121-125. we will not explore RF-C in greater depth. protein-protein interactions The catalytic subunit has 5’→ 3’ DNA polymerase activity and an intrinsic 3'→ 5' exonuclease activity.S. X. (1994) Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA.. Burgers. Because we have already discussed the γ complex in some detail. The matchmaker in this case is an associated protein complex known as replication factor C (RFC). P. one goal.. Addition of the 66 kDa subunit appears to result in dimerization of the tetrameric complex. the clamp does not interact directly with the catalytic subunit. (Krishna. similar to Pol III holoenzyme. and O’Donnell. and T7 processivity factors. PCNA requires a clamp loader to become associated with the template primer.. The only major structural difference is that PCNA and gp45 are homotrimers.
RF-C and PCNA are regarded as pol accessory factors: e. The human pol has 4 subunits: 260 kDa-. the POL3 gene (which encodes the catalytic subunit) is essential for progression through the cell cycle. protein-protein interactions 12 kDa-.000 amino acid extension that is unique to Pol ε . the primary function of Pol δ is to elongate RNA/DNA primers laid down by Pol α (original observation from the SV40 system). Pol ε is a reasonably processive enzyme that also associates with PCNA at a primer terminus. These proteins are not expressed in quiescent cells (G0).
. Biochemical and genetic studies in yeast and mammalian cells suggest that Pol δ is also involved in some types of DNA repair synthesis. Pol δ. In vivo. There is much evidence that Pol δ is essential for replication.structural.pol catalytic subunit 59 kDa-. PCNA and Pol δ.It is worth noting that in eukaryotic systems. The N-terminal portion contains the catalytically important residues. DNA polymerase ε Pol ε is another multisubunit nuclear DNA polymerase. some of which are involved in cell cycle regulation (check point control). • Pol δ and PCNA levels are increased in proliferating cells (generally parallel Pol α levels). It also has 3' →5' exonuclease activity. This domain is known to interact with regulatory proteins. pol3 ts mutants are blocked in DNA synthesis and arrest in S-phase at non-permissive temperature. for example: • In yeast. This probably has more to do with the fact that the Pol III holoenzyme components are more tightly associated and can more easily be co-purified. coli) the γ complex and β are considered to be part of Pol III holoenzyme.structural. • Complete SV40 DNA replication in vitro requires RF-C.g. By contrast. in prokaryotes (E. expression of these proteins is increased at the G1/S boundary. because Pol ε does not appear to be greatly stimulated by PCNA. RF-C and PCNA are sometimes referred to as the Pol δ complex. In yeast. protein-protein interactions The Pol ε catalytic subunit is one of the largest polymerase activities yet described. Physiological role of Pol δ. The significance of this is not clear. The C-terminal part consists of a 1. than to any significant functional difference.multimerization (?) 17 kDa-.
suggesting that it is the C-terminal region of the protein which supplies an essential. Unlike Pol δ. Pol β expression is considered constitutive. indicating that Pol β is not essential for replicative synthesis. Pol ε is not required for complete SV40 replication in vitro and it is not an effective substitute for Pol δ in this system. Clearly. Pol β levels increase following treatment of cells with agents that damage DNA. Yeast pol4 mutants are viable. Pol δ and Pol ε activities are not equivalent. Biochemical and genetic studies in yeast suggest that Pol ε is also involved in some DNA repair synthesis. The mutants are. It is possible that these polymerases have different. non-redundant function. Pol ε has been detected at the replication fork (along with Pol α and Pol δ) in mammalian cell extracts. In support of this. is essential for cell viability and DNA replication in yeast. Interestingly. in yeast. or is involved in quality control/ cell cycle control. It is necessary for the completion of S phase. although it can bind a nicked duplex and is capable of some limited displacement synthesis. sensitive to certain mutagenic agents. it may also associate with PCNA. even in rapidly dividing cells. Perhaps Pol ε plays some organizational role. again. it is composed of a single ~40-48 kDa protein. All of these observations suggest that Pol β is primarily involved in DNA repair. DNA polymerase β This is the smallest and simplest of the classical eukaryotic polymerases. It is also expressed in quiescent cells. However. Pol β is not highly active and is not very processive. Further. it has been further suggested that Pol ε is involved in primer elongation. Because of its high processivity and proofreading activity.
. specialized roles in at the replication fork. This suggests that it is a replicative polymerase. Its preferred template is duplex DNA with short gaps. in addition to Pol δ. The role of this enzyme in DNA replication is controversial. In yeast. It has no intrinsic exonuclease activities.The physiological role of Pol ε . Like most single polypeptide enzymes. Other evidence argues against a major role for Pol ε in bulk DNA synthesis. the N-terminal mutants accumulate DNA damage and show defects in cell cycle regulation of DNA synthesis. deletion mutants lacking the entire N-terminal DNA polymerase domain of Pol ε are viable. However. Pol β is encoded by POL4. however. Expression levels are low and remain constant throughout the cell cycle. the gene which encodes the large catalytic subunit. Genetic analysis in yeast has revealed that POL2.
RF-C and PCNA probably prevent continued (unedited) synthesis of DNA by Pol α and limit its activity to a few incorporated resides. Using viral DNA template (5 kb circular dsDNA. S. DNA ligase I (Okazaki fragment processing) RPA (ssDNA binding protein) DNA topoisomerase I and II (conformational issues) SV40 T-antigen (initiation protein and helicase) For our purposes at this time. with its associated primase activity and lower processivity. is ideally suited for priming both leading and lagging strand synthesis. which functions as an initiation protein and a helicase) it has been possible to achieve complete replication with highly purified cellular proteins. and possibly asymmetric. By analogy with Pol III. Priming of the leading strand occurs only once at each replication fork. Recent evidence indicates that Pol δ is probably dimeric. and Stillman. The product of Pol α :primase action is iRNA/iDNA.) Pol δ then associates with the PCNA clamp and the primer terminus and begins processive extension of the primer until the Okazaki fragment is completed. Lagging strand synthesis requires repeated initiation. (Waga. displacing Pol α :primase. PCNA. 31) Pol α . RF-C (Pol δ complex) FEN1 (MF-1). we will focus on the polymerases. or plasmids containing a viral origin of replication) and a single viral protein (T-antigen. RF-C (clamp loader) associates with the 3'OH terminus of the DNA primer and loads a PCNA clamp on the template-primer.. B. This would help to coordinate leading
. two δ pols may simultaneously replicate the leading and the lagging strand. (1994) Anatomy of a DNA replication fork revealed by reconstitution of SV40 replication in vitro. RNase H.Polymerase switching in eukaryotic DNA replication Studies that have defined the replication fork in eukaryotes have employed the SV40 system. each Okazaki fragment requires priming. Nature 369: 207-212. (RNA-DNA primers syntesized are shorter if RF-C and Pol δ are present.) The current picture of the eukaryotic replication fork (with respect to polymerase activities) is as follows: (Fig. The cellular proteins/complexes required are: Pol α :primase Pol δ.
We will not discuss RNA viruses and RdRPs further. Cell 78: 725-728). influenza virus. and adenovirus. (1994) Smart machines at the DNA replication fork. In any case. (For review see: Stillman. Examples include herpesvirus.and lagging strand synthesis. poliovirus. Cellular RdRPs also exist. it is useful to look at a few interesting viral systems. • Viruses that have genomes composed of RNA and do not use DNA polymerases during replication (eg. These agents use a virus coded RNA dependent DNA polymerase (reverse transcriptase) and host DNA-dependent RNA polymerase to replicate their genomes. Pol α :primase may preferentially associate with the Pol δ activity replicating the lagging strand. plant geminiviruses). Examples include avian sarcoma virus (ASV). from Pol α :primase to Pol δ. both leading and lagging strand synthesis requires a polymerase switch. These viruses encode RNA-dependent RNA polymerases (RdRP). We will also discuss a unique and essential cellular polymerase that is specialized to function at chromosome ends. SV40 and polyoma virus in mammals. • Medium to large viruses that encode their own DNA polymerases and accessory proteins. tobacco mosaic virus and most plant viruses. although this is not clear.
. poxvirus. • Viruses that encapsidate their genome as RNA but replicate it through a DNA intermediate (retroviruses). Other eukaryotic DNA polymerases Now that we have learned about the eukaryotic nuclear polymerases. Pol ε may also elongate some RNA-DNA primers in vivo. Pol δ elongates both the leading strand primer and lagging strand primers in the SV40 system. B. and human T cell lymphotrophic virus (HTLV). Eukaryotic viruses can be placed into four broad groups on the basis of polymerase activities used during replication. phage Qβ). these are involved in gene regulatory pathways (such as gene silencing) that utilize small RNA molecules (microRNAs and small interfering RNAs) to degrade mRNAs in a sequencespecific manner.g. We will briefly talk about herpesvirus and retrovirus systems. human immunodeficiency virus (HIV). • Small viruses that rely on host DNA polymerases to replicate their genomes (e.
systemic neonatal infections). it strongly binds dsDNA in a sequence nonspecific manner. That is. a protein with strong affinity for dsDNA. Hodgkin’s and Burkitt's lymphoma. retinitis. because by itself UL42 has a strong affinity for dsDNA. UL42 serves anchors the polymerase to the template. holding UL42 in close proximity to the DNA without preventing its diffusion along the DNA backbone. However. However. Some examples include herpes simplex virus (cold sores. HSV) is replicated by viral DNA polymerase with the aid of six virus coded accessory proteins. while thioredoxin does not. wasting disease. UL42 may work like an electrostatic
. The pol subunit has an intrinsic 3' → 5' exonuclease activity. Rather. The pol/UL42 polymerase holoenzyme is highly processive. genital lesions. • UL42 does not form a ring around the DNA. and Epstein-Barr virus (infectious mononucleosis. it structurally resembles a partial PCNA ring. sporadic encephalitis.Herpesvirus DNA polymerase Herpesviruses infect a variety of animal species. and plasmids expressing viral replication proteins. UL42 is a novel type of clamp. which is the catalytic subunit (pol). • UL42 doesn't require a clamp loader or ATP to be loaded on the template primer. nasopharyngeal carcinoma). positively charged surface of UL42 and the negatively charged DNA acts a non-specific tether. and it is UL42 that serves as the processivity factor. varicella zoster virus (chicken pox. The viral DNA polymerase exists as a heterodimer of UL30 (140 kDa). cytomegalovirus (deafness and retardation in fetus. it appears that the high affinity of UL42 for dsDNA and pol (which it binds simultaneously) increases the affinity of holoenzyme for the template primer. but mostly mammals and birds. The large herpesvirus genome (~153 kbp. The mechanism also differs from the thioredoxin clamp of T7 polymerase. and UL42 (52 kDa). But recent structural data suggests that electrostatic attraction between a large. How this can occur without slowing elongation is not entirely clear. A model system to study herpesvirus replication consists of cells transfected with a plasmid containing a viral origin of replication. shingles). The mechanism differs from that of the β subunit of Pol III and PCNA in several respects: • UL42 has a high intrinsic affinity for DNA. fever blisters. pneumonia in immunocompromised adults). In short.
g. (Actually. Ann.
. But only the HSV pol is sensitive to phosphonoacetic acid. J.J. viruses are also valuable model systems for the analysis of cellular replication mechanisms. Both host polymerases and HSV pol are sensitive to the nucleoside analogue aphidicolin.. Filman. We will use herpesviruses as an example of how selective inhibition can be achieved. P. (2000) The crystal structure of an unusual processivity factor. is less toxic and has some clinical value. Interference with viral replication protein functions/interactions.e.J. activated) only in infected cells. H. thymidine kinase (TK).M. Molecular Cell 5: 267-278.. it turns out that all of the essential herpesvirus replication proteins directly or indirectly interact with each other. The most effective selective inhibitor of this type is the nucleoside analogue acyclovir (acycloguanosine). Sensitivity of polymerase to inhibitors. (Zuccola. and cellular enzymes convert it to triphosphate). D. Biochem. and is one of the major goals of researchers studying how viruses replicate. phosphonoacetic acid is toxic and drug resistant mutants emerge rapidly. phosphonoformic acid.. which acts as a chain terminator. As a result. D. herpes virus UL42.“tractor field”. 32).E. Of course. pol:UL42).) (For a review of herpesvirus replication see: Boehmer. Another way to inhibit virus replication is to employ compounds that interfere with the interaction of viral replication proteins (e. which inhibits release of pyrophosphate (Fig. Such inhibitors are not yet available. HSV TK converts acyclovir to a monophosphate. that is. Unfortunately. and how the proteins within them interact. Herpesvirus DNA polymerase can be distinguished from host polymerases by its sensitivity to inhibitors. (1997) Herpes simplex virus DNA replication. Rev. I. Knowledge of the functions of viral replication complexes. acyclovir is converted into the triphosphate form (i. and Hogle. and Lehman. but not host. will eventually permit the design of drugs (small molecules) that selectively interfere with these interactions. Actually. Obviously. 66: 347-384. bound to the C-terminus of its cognate polymerase. Target cell approach Another way of controlling virus infections is the target cell approach. selective inhibition of viral polymerase is an attractive method for controlling virus infections. A derivative.R. thereby blocking virus replication. to use drugs that can only enter or be activated in infected cells..) Controlling virus infections Selective inhibition of virus replication is an attractive means of controlling virus infections. Acyclovir is an effective agent against some herpesviruses because it is a substrate for viral. Coen.
Retrovirus reverse transcriptase Retroviruses package their genomes as ssRNA but replicate this RNA through a dsDNA intermediate. and therefore has no proofreading function.
. So. As a result. hepatitis B virus (hepadnavirus) and cauliflower mosaic virus. For example.TK and HSV-TK Thymidine → TMP → TTP
HSV-TK Acyclovir → Acyclovir-MP
Acyclovir has therapeutic value only against herpesviruses like HSV and varicella-zoster which encode a kinase that can convert acyclovir to an activated substrate for DNA polymerase. Reverse transcription is part of the transposition process in these elements. they employ an RNA dependent DNA polymerase (reverse transcriptase). In addition. virus variants are generated at high frequency. its error rate is relatively high. This is an important aspect of pathogenesis: retroviruses are adept at evading host immunosurveillance because of high mutation frequency. Examples of important human retroviruses include human T cell lymphotrophic virus (HTLV) and human immunodeficiency virus (HIV). it is not a substrate for 3'→ 5' exonuclease and so is not removed from the 3' terminus. some of which resemble retroviruses in their structure and organization. and copia and P elements in Drosophila. It has no activity against cytomegalovirus (CMV). and requirements for dNTPs and Mg . To do this. Examples include Ty in yeast. This is reflected in a high mutation rate. Retroviruses primarily infect mammals and birds. RT does not have a detectable DNA specific exonuclease activity. Also. requirement for a template primer with a 3'-OH terminus. information flow from RNA to DNA appears to occur in many systems. RT is a typical DNA polymerase in the 5'→ 3' direction of synthesis. some types of cellular transposons (retroposons) also encode reverse transcriptase. Acyclovir triphosphate is used as a substrate in place of dGTP. Properties of reverse transcriptase. It is a much more potent inhibitor of viral polymerase than it is of cellular polymerases. When it is incorporated into DNA it acts as a chain terminator. Reverse transcriptases are also used by related viruses that package DNA in their capsids and replicate through RNA intermediates. which does not encode a TK.
as well as palm. It can also act as an endonuclease.A. and RNase H activities of the retrovirus reverse transcriptase. and Steitz. • It has intrinsic RNase H activity. The first step in replication involves synthesis of an ssDNA copy (cDNA) of the genomic viral RNA (Fig. Avian myeloblastosis virus (AMV) RT is well studied and is often used in the lab. for obvious medical reasons. C.. and appears to function in initiation. HIV RT is also well studied. In particular. as well as transcription by host RNA polymerase II (a DNA dependent RNA polymerase). Note that retroviral replication requires both the RNA dependent and DNA dependent DNA polymerase. Ann. Note that the replication mechanism requires RT to use both an RNA and a DNA template. finger. 34). The product is a DNA-RNA hybrid. T. In the next step. and is primed by a host tRNA which hybridizes to the template RNA.
. The β subunit (63-66 kDa) has polymerase activity and RNase H activity. and shows remarkable structural homology to Pol I. yields a duplex DNA. RNase H hydrolyzes phosphodiester bonds to leave products with 3' hydroxyl and 5' phosphate ends.M. The enzyme is relatively processive and can replicate the 8 kb retrovirus genome without a processivity factor.) Retrovirus replication A simplistic picture of the retrovirus replication cycle is presented here.RT is unique among DNA polymerases in at least two respects: • It can use primed. Biochem. and second strand DNA synthesis. RNase H is a processive exonuclease that specifically degrades the RNA strand of a DNA-RNA hybrid beginning from either the 5' or 3' end. Its crystal structure has been solved. It can also use a primed ssDNA as template. it has a similar cleft. the RNA is degraded by RNase H. The α subunit (51 kDa) has neither activity. The retrovirus RNA genome is typically about 8 kb in length. Synthesis is accomplished by reverse transcriptase. also catalyzed by reverse transcriptase. Rev. natural ssRNAs as template. and thumb domains (Fig. (1994) Function and structure relationships in DNA polymerases. 33) (For review see: Joyce. 63: 777-822. The RT holoenzyme is a dimer of two related proteins. primarily for the synthesis of cDNA.
where its genes are transcribed by host RNA polymerase II to generate mRNAs encoding viral proteins.) Full-length transcripts generated from the provirus are assembled into virus particles to complete the replication cycle. One strand. the 3' end of the newly synthesized chromosome can't be copied. The integrated copy of the viral genome is called a provirus. The number of repeats is somewhat variable. The number of repeats can vary widely between species. contains clusters of G-residues. However. because there is no primer available. If not corrected. the virus is essentially invisible to the hosts’ immune system. Biochem. (1996) Telomere length regulation. Each species has a characteristic telomeric DNA sequence. They are an essential feature of linear chromosomes that "seal" the chromosome end and prevent the loss of terminal DNA sequences. Consider: following replication. 65: 337-365. although the same sequence can occur in more than one species. 35). The RNA component
.W. however.The duplex DNA is integrated into the host cell genome (catalyzed by a viral integrase). it synthesizes DNA using an RNA template. but is usually maintained within relatively narrow limits within a species. This problem is avoided by the de novo addition of telomeric sequence by telomerase. 2) specialized proteins that interact with the DNA sequences. Telomeres and Telomerase Telomeres are specialized structures found at the ends of linear. C. Ann. Rev. The protein and RNA components make up an active enzyme of ~200 kDa. the one found at the 3' end of each chromosomal DNA strand. eukaryotic chromosomes. 1) short DNA sequences (typically 6 to 26 nt long) that are repeated many times.) Telomerase This enzyme is a specialized reverse transcriptase: that is. it is a ribonucleoprotein. for example: Tetrahymena Human Yeast TTTTGGGG TTAGGGG TGTGGGTGTG
The lagging strand problem Why are telomere repeats needed? Because of the 3' end problem (Fig. Telomeres have two components. unlike conventional reverse transcriptase. this would result in the loss of sequences following each chromosome duplication. (For review see: Greider. (In this form.
How does telomerase work? In vitro. to yield: TTGGGG(TTGGGG)n. Because the telomere sequence is repeated. The proposed mechanism by which telomerase adds to the 3’ end is as follows: Using a short. Introducing a change in the sequence of the TLC1 gene (within the RNA sequence used as template) results in that change being introduced into telomeres: e. TGTGGGTGTG to TGTGTGGGCCTG. In yeast and ciliated protozoa like Tetrahymena. but its activity appears to negatively regulated by other proteins.3 kb in yeast) contains the template sequence that is used for DNA synthesis. pombe also results in telomere shortening and rapid senescence. Tetrahymena telomerase will repeatedly add the same telomere sequence to the 3' end. and mammals.
. cerevisiae. the enzyme adds nucleotides (the telomeric repeat). Disruption of this gene (TLC1 in yeast) results in progressive shortening of telomeres by several bp/generation. So.(~1. Gene disruption in S. given the oligonucleotide TTGGGG. telomerase from a given species synthesizes the G-rich strand sequence characteristic of the species. fission yeast. and human. yeast. including ciliates (Tetrahymena and Euplotes). This allows the 5' end to be lengthened by conventional priming and DNA synthesis. For example.g.) Genes encoding the RNA subunit have been isolated and cloned from several sources. In S. 36). All that is required is a telomere DNA sequence primer and the appropriate dNTPs. the catalytic subunit corresponds to the Est2 gene (ever shorter telomeres). it doesn't really matter what the exact number of telomere repeats is. as long as there are enough. where n can be >100. Euplotes. and the mechanism may vary between organisms. (So telomerase carries its own template. How is telomere length controlled? This is not entirely clear. then translocates and repeats the process (Fig. There appears to be two protein subunits. a large catalytic subunit (103-127 kDa) and a small subunit of about 43 kDa. the strategy used by the cell to avoid the 3' end problem is to increase the length of the 3' end of the chromosome. telomerase is expressed constitutively. complementary sequence in its RNA component as template. The protein subunits of telomerase have been identified and cloned from budding yeast. Disruption of genes encoding these regulatory proteins (in yeast) results in massive telomere elongation. The large subunit shares important sequence motifs with HIV RT and other DNA polymerases.
including immortalized (transformed) cells in culture. However. telomerase activity is present in actively dividing cells. So telomerase may be useful for cancer diagnostics. in differentiated (quiescent) human somatic cells (which start with ~10 kb of telomerase sequence at each chromosome end).In contrast. and is a possible target for therapeutics. telomerase activity can't be detected and telomeres shorten progressively with age and with each cell division (a marker for cell age?). and in most cancer cells.