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A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand.

DNA polymerases are best-known for their role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand. This process copies a piece of DNA. The newlypolymerized molecule is complementary to the template strand and identical to the template s ori!inal partner strand. DNA polymerases use a ma!nesium ion for catalytic acti"ity.

Function
DNA polymerase can add free nucleotides to only the # end of the newly-formin! strand. This results in elon!ation of the new strand in a $ -# direction. No known DNA polymerase is able to be!in a new chain %de novo&. DNA polymerase can add a nucleotide onto only a preexistin! # -'( !roup, and, therefore, needs a primer at which it can add the first nucleotide. )rimers consist of *NA and DNA bases with the first two bases always bein! *NA, and are synthesized by another enzyme called primase. An enzyme known as a helicase is re+uired to unwind DNA from a double-strand structure to a sin!le-strand structure to facilitate replication of each strand consistent with the semiconser"ati"e model of DNA replication. ,rror correction is a property of some, but not all, DNA polymerases. This process corrects mistakes in newly-synthesized DNA. -hen an incorrect base pair is reco!nized,

DNA polymerase re"erses its direction by one base pair of DNA. The # -$ exonuclease acti"ity of the enzyme allows the incorrect base pair to be excised %this acti"ity is known as proofreading&. .ollowin! base excision, the polymerase can re-insert the correct base and replication can continue. /arious DNA polymerases are extensi"ely used in molecular biolo!y experiments.

Variation across species


DNA polymerases ha"e hi!hly-conser"ed structure, which means that their o"erall catalytic subunits "ary, on a whole, "ery little from species to species. 0onser"ed structures usually indicate important, irreplicable functions of the cell, the maintenance of which pro"ides e"olutionary ad"anta!es. 1ome "iruses also encode special DNA polymerases, such as (epatitis 2 "irus DNA polymerase. These may selecti"ely replicate "iral DNA throu!h a "ariety of mechanisms. *etro"iruses encode an unusual DNA polymerase called re"erse transcriptase, which is an *NA-dependent DNA polymerase %*dDp&. 3t polymerizes DNA from a template of *NA.

DNA polymerase families


2ased on se+uence homolo!y, DNA polymerases can be further subdi"ided into se"en different families4 A, 2, 0, D, 5, 6, and *T.

Family A
)olymerases contain both replicati"e and repair polymerases. *eplicati"e members from this family include the extensi"ely-studied T7 DNA polymerase, as well as the eukaryotic mitochondrial DNA )olymerase 8. Amon! the repair polymerases are Escherichia coli DNA pol 3, Thermus aquaticus pol 3, and Bacillus stearothermophilus pol 3. These repair polymerases are in"ol"ed in excision repair and processin! of 'kazaki fra!ments !enerated durin! la!!in! strand synthesis.

Family B
)olymerases mostly contain replicati"e polymerases and include the ma9or eukaryotic DNA polymerases :, ;, <, %see =reek letters& and also DNA polymerase >. .amily 2 also includes DNA polymerases encoded by some bacteria and bacteriopha!es, of which the best-characterized are from T?, )hi@A, and *2BA bacteriopha!es. These enzymes are in"ol"ed in both leadin! and la!!in! strand synthesis durin! replication. A hallmark of the 2 family of polymerases is their hi!hly faithful DNA synthesis durin! replication. -hile many ha"e an intrinsic # -$ proofreadin! exonuclease acti"ity, eukaryotic DNA polymerases : and > are two examples of 2 family polymerases lackin! this proofreadin! acti"ity.

Family C
)olymerases are the primary bacterial chromosomal replicati"e enzymes. DNA )olymerase 333 alpha subunit from E. coli is the catalytic subunit CDE and possesses no known nuclease acti"ity. A separate subunit, the epsilon subunit, possesses the # -$ exonuclease acti"ity used for editin! durin! chromosomal replication. *ecent research has classified .amily 0 polymerases as a subcate!ory of .amily 5.

Family D
)olymerases are still not "ery well characterized. All known examples are found in the ,uryarchaeota subdomain of Archaea and are thou!ht to be replicati"e polymerases.

Family X
0ontains the well-known eukaryotic polymerase pol F, as well as other eukaryotic polymerases such as pol G, pol H, pol I, and terminal deoxynucleotidyl transferase %TdT&. )ol F is re+uired for short-patch base excision repair, a DNA repair pathway that is essential for repairin! abasic sites. )ol H and )ol I are in"ol"ed in non-homolo!ous end9oinin!, a mechanism for re9oinin! DNA double-strand breaks. TdT is expressed only in lymphoid tissue, and adds "n nucleotides" to double-strand breaks formed durin! /%D&J recombination to promote immunolo!ical di"ersity. The yeast Saccharomyces cerevisiae has only one )ol 5 polymerase, )ol?, which is in"ol"ed in non-homolo!ous end-9oinin!.

Family Y
)olymerases differ from others in ha"in! a low fidelity on undama!ed templates and in their ability to replicate throu!h dama!ed DNA. Kembers of this family are hence called translesion synthesis %TL1& polymerases.

Family RT
The re"erse transcriptase family contains examples from both retro"iruses and eukaryotic polymerases. The eukaryotic polymerases are usually restricted to telomerases. These polymerases use an *NA template to synthesize the DNA strand.

Prokaryotic DNA polymerases


2acteria ha"e $ known DNA polymerases4

Pol I4 implicated in DNA repairM has $ -N# %)olymerase& acti"ity and both # -N$ exonuclease %)roofreadin!& and $ -N# exonuclease acti"ity %*NA )rimer remo"al&. Pol II4 in"ol"ed in reparation of dama!ed DNAM has # -N$ exonuclease acti"ity.

Pol III4 the main polymerase in bacteria %elon!ates in DNA replication&M has # N$ exonuclease proofreadin! ability. Pol IV4 a 6-family DNA polymerase. Pol V4 a 6-family DNA polymeraseM participates in bypassin! DNA dama!e.

Substrate requirements for synthesis of new DNA


DNA polymerase 3 catalyzes the addition of a complementary dNT) to the # '( end of a polydeoxynucleotide chain. The reaction mechanism is a simple nucleophilic displacement. There are .'O* essential re+uirements for the acti"ity of DNA polymerase 34 1 T!"P#AT!

There must be a template strand to be copied. $ PRI"!R DNA polymerase 3 %and e"ery other known DNA polymerase& cannot initiate DNA synthesis by itself. 3t can only extend a pre-existin! DNA chain. % %&'( !ND The reaction mechanism re+uires that the primer must ha"e a free # '( end for synthesis to continue.
The Sanger method of DNA sequencing actually makes use of this requirement by using low concentrations of dideoxy nucleoside triphosphates (ddNTPs in a DNA synthesis reaction! ddNTPs are missing the "#$% group! $nce a ddNTP is incorporated into a DNA chain& synthesis will stop!

) *eo+ynucleosi*e trip,osp,ates dNT)s must be present - usually as the K!PP salt.

Energetics of DNA synthesis


.a"ourable ener!etic factors are4 (ydrolysis of the pyrophosphate that is released. .ormation of hydro!en bonds and base-stackin! interactions.

Onfa"ourable ener!etic factors are4

The phosphoric anhydride bond in the dNT) is replaced with a phosphodiester bond in the polynucleotide. The ener!y balance of this chan!e is sli!htly unfa"ourable. Loss of entropy as the free dNT) is incorporated into the polymer.

Continuity of DNA synthesis 3n thinkin! about the mechanism of synthesis of DNA polymerase 3 %or any DNA polymerase, for that matter&, we must also consider what happens after each dNT) has been incorporated.There are two possibilities4 After eac, nucleoti*e a**ition- t,e en.yme/template/primer comple+ *issociates. A new complex will need to form between enzyme, template and primer - with the # '( end properly positioned for catalysis - before the next dNT) can associated and be 9oined to the !rowin! polynucleotide chain. This mode of synthesis is called *istri0uti1e synt,esis. After eac, a**ition- t,e en.yme mo1es or sli*es one place alon2 t,e template/primer. 3n this case, the enzyme can be "iewed as slidin! alon! the DNA after each dNT) has been incorporated. This mode of synthesis is called processi1e synt,esis. 3n reality, the processi"ity or distributi"ity of any DNA polymerase is an intrinsic property of each enzyme or enzyme complex and it will "ary for each case. No enzyme is completely processi"e and none is completely distributi"e. The len!th of 'kazaki fra!ments depend primarily upon the processi"ity of the correspondin! DNA polymerase. DNA polymerase I DNA polymerase 3 is a A@Q amino-acid polypeptide %K-RDS#DDQ& encoded by the polA !ene. 3t has three distinct enzymatic acti"ities4

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A $ -N # polymerase acti"ity A # -N $ exonuclease acti"ity A $ -N # exonuclease acti"ity

1tructurally, the enzyme has two domains which can be separated by proteolysis with trypsin or subtilisin, both of which clea"e at the amino acid #@?. The smaller %#? kD& Nterminal fra!ment carries the $ -N # exonuclease acti"ity. The lar!er %7B kD& 0-terminal fra!ment carries the polymerase and # -N $ exonuclease acti"ities. This fra!ment is often called 3leno4 fra2ment after (ans 3leno4 who first disco"ered this proteolytic clea"a!e. Tlenow fra!ment is the most commonly used DNA polymerase in molecular biolo!y labs %at least until Ta+ polymerase came alon!U&. 3t is used in reactions to label DNA fra!ments for hybridization analyses. The # -N $ exonuclease acti"ity of DNA polymerase is not simply due to the catalysis of the re"erse polymerase reaction but is a separate and distinct enzymatic acti"ity. The function of the # -N $ exonuclease acti"ity is that of PR''F/R!ADIN5. Any nucleotides -- such as transiently pairin! tautomers -- that are incorrectly incorporated are excised by this acti"ity. DNA polymerase I is not the replicative polymerase Ontil DABA, DNA polymerase 3 was the only known DNA polymerase in E. coli. (owe"er, as DNA polymerase 3 was characterized further, it became clear that the properties of this enzyme were not suitable for an enzyme with a central role in DNA replication4 The enzyme is too slow! DNA polymerase 3 catalyzes the incorporation of dNT)s at a maximal rate of @S ntVsec %approximately&. At this rate, it would re+uire approximately ?BS,SSS sec %R 7BB7 min R D@Q hr R $.# days& to replicate the entire E. coli chromosomeU This is much too slow for an or!anism which can di"ide e"ery @S mins. The enzyme is too abundant There are approximately ?SS molecules of DNA polymerase 3 per E. coli cell. This is excessi"e !i"en that there are !enerally only @ replication forks per cell. The enzyme is not processive enough DNA polymerase 3 dissociates after catalysin! the incorporation of @S-$S nucleotides. DNA polymerase I cannot initiate DNA synthesis de novo. (owe"er, it must be noted that DNA polymerase 3 is not uni+ue in this re!ard. 3t shares this particular problem with e"ery other known DNA polymerase. Cells containing the polA1 mutation are viable. 3n DABA, De Luca and 0airns reported the isolation of a mutant of E. coli that was "iable e"en thou!h it lacked DNA polymerase 3 acti"ity. The mutant was found

to be an amber mutantM we now know from se+uence information that the codon for tryptop,an %)$ is mutated to a stop codon in this mutant. 0ells carryin! this mutation !row at normal rates. (owe"er, they are more sensiti"e than normal to muta!enic a!ents such as O/ li!ht. DNA polymerase II This enzyme is most likely in"ol"ed in DNA repair systems. The enzyme is QAA@D kD in size and is coded by the polB !ene. 1trains lackin! the !ene show no defect in !rowth or replication. 1ynthesis of PolII is induced durin! the stationary phase of cell !rowth. This is a phase in which little !rowth and DNA synthesis occurs. 3t is also a phase in which the DNA can accumulate dama!e such as short !aps, which act as a block to PolIII. Onder these circumstances, PolII helps to o"ercome the problem because it can reinitiate DNA synthesis downstream of !aps. PolII has a low error rate but it is much too slow to be of any use in normal DNA synthesis. DNA polymerase III The DNA polymerase 333 holoenzyme is the principal replicati"e enzyme in E. coli. This enzyme is hi!hly processi"e and catalyses polymerization at a hi!h rate. The enzyme is a complex of DS polypeptides.There are two forms of the enzyme.

Core en.yme
The core enzyme consists of only those subunits that are re+uired for the basic underlyin! enzymatic acti"ity. 3n the case of DNA polymerase 333, the core enzyme consists of three subunits4 alpha % &, epsilon % & and theta % &.

The alpha % & subunit, which is coded by the polC !ene, is a D@AAS? kD polypeptide that contains the $ -N # DNA polymerase acti"ity but no proofreadin! a"ti"ity. The epsilon % & subunit, which is coded by the dnaQ !ene is a @7SAA kD polypeptide that contains the proofreadin! # -N $ exonuclease acti"ity. The function of the QQ?B kD theta % & subunit, which is encoded by the holE !ene is not known. 3t is not essential for !rowth and is !enerally not conser"ed in bacteria.

Althou!h the core enzyme can catalyse DNA synthesis, it is not processi"e -- only about DS -D$ nt are incorporated at a time.

(oloen.yme

The holoenzyme is the fully functional form of an enzyme, complete with all of its necessary accessory subunits. The DNA polymerase 333 holoenzyme consists of the core enzyme %described abo"e&, the slidin! clamp and the clamp-loadin! complex.

The sliding clamp The beta % & subunit is essential for processi"ity - as lon! as it is present, the enzyme has
almost unlimited processi"ity. This ?S.B kD polypeptide, coded by dnaN, has been crystallized. 3t functions as a slidin! clamp . 3t assembles into a dimer with a circular structure throu!h which a DNA double helix can pass.

The clamp-loading comple


The clamp-loadin! complex consists of the delta % & %#Q.7 kD& and delta % && prime %#B.A kD&, chi % & %DB.B kD& and psi % & %D$.@ kD& subunits and either or both of the !amma % & subunit %BQ.? kD& and the tau % & subunit %7D.D kD&. 2oth the !amma and tau subunits are encoded by tthe dnaX !ene. This !ene is translated in two different ways. 3f the !ene is translated completely then the tau % & subunit is synthesized. 3f translation slips or frameshifts part way throu!h the readin! frame then a stop codon is encountered, and the !amma % & subunit is synthesized. 2oth the !amma % & subunit and the tau% & subunit are motor AT)ases. The structures of many of these subunits ha"e now been sol"ed and they are be!innin! to re"eal how the different subunits interact and function in the buildin! of a replisome and in its on!oin! function. The subunit binds to the subunit and, in concert with the and subunits and with AT), it catalyses the openin! of the dimer to permit passa!e of DNA. DNA polymerase III ,oloen.yme is the primary enzyme complex in"ol"ed in prokaryotic DNA replication. 3t was disco"ered by Thomas Tornber! and Kalcolm =efter in DA7S. The complex has hi!h processi"ity %i.e. the number of nucleotides added per bindin! e"ent& and, specifically referrin! to the replication of the E.coli !enome, works in con9unction with four other DNA polymerases %)ol 3, )ol 33, )ol 3/, and )ol /&. 2ein! the primary holoenzyme in"ol"ed in replication acti"ity, the DNA )ol 333 holoenzyme also has proofreadin! capabilities that correct replication mistakes by means of exonuclease acti"ity workin! # -N$ . DNA )ol 333 is a component of the replisome, which is located at the replication fork.

Components
The replisome is composed of the followin!4

@ DNA Pol III en.ymes, made up of 6, 7 and 8 subunits. o the : subunit has polymerization acti"ity.

the < subunit has proofreadin! acti"ity. the W subunit stimulates the < subunit s proofreadin!. @ 9 units which act as slidin! DNA clamps, they keep the polymerase bound to the DNA. @ : units which connect the @ DNA )ol 333 enzymes. D ; unit which acts as a clamp loader for the la!!in! strand 'kazaki fra!ments, helpin! the two F subunits to form a unit and bind to DNA. The 8 unit is made up of $ 8 subunits.
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Acti1ity
DNA polymerase 333 acti"ity be!ins after strand separation at the ori!in of replication. 2ecause DNA synthesis cannot start de novo, an *NA primer, complementary to part of the sin!le-stranded DNA, is synthesized by primase %an *NA polymerase&4 %"U" for *NA, "X" for DNA, "Y" for polymerase&
--------> ! _ G * $ _ ( ! _ U * A _ ( ! _ A * % _ ( ! _ U * A _ ( * * * * _ _ _ _ | RNA | <--ribose (sugar)-phosphate backbone | Po | <--RNA pri!er |_ _ _ _| <--h"#rogen bon#ing G $ A % $ $ <--te!p ate ss&NA (sing e-stran#e# &NA) _ _ _ _ _ _ <--#eo'"ribose (sugar)-phosphate backbone ( ( ( ( ( (

A**ition onto %&'(


As replication pro!resses and the replisome mo"es forward, DNA polymerase 333 arri"es at the *NA primer and be!ins replicatin! the DNA, addin! onto the # '( of the primer4
! _ G * $ _ ( ! _ U * A _ ( ! _ A * % _ ( ! _ U * A _ ( * * * * _ _ _ _ | &NA | <--ribose (sugar)-phosphate backbone | Po | <--RNA pri!er |_)))_ _| <--h"#rogen bon#ing G $ A % $ $ <--te!p ate ss&NA (sing e-stran#e# &NA) _ _ _ _ _ _ <--#eo'"ribose (sugar)-phosphate backbone ( ( ( ( ( (

<ynt,esis of DNA
DNA polymerase 333 will then synthesize a continuous or discontinuous strand of DNA, dependin! if this is occurrin! on the leadin! or la!!in! strand %'kazaki fra!ment& of the DNA. DNA polymerase 333 has a hi!h processi"ity and therefore, synthesizes DNA "ery +uickly. This hi!h processi"ity is due in part to the F-clamps that "hold" onto the DNA strands.

-----------> ! _ G * $ _ ( ! _ U * A _ ( ! _ A * % _ ( ! _ U * A _ ( ( _ $ * G _ ( ( _ G * $ _ ( ( _ % * A _ ( ( _ A * % _ ( ( _ G * $ _ (

* * * * ( _ _ _ _ _| &NA | <--#eo'"ribose (sugar)-phosphate backbone G| Po | <--RNA pri!er *|_)))_ _| <--h"#rogen bon#ing $ <--te!p ate ss&NA (sing e-stran#e# &NA) _ <--#eo'"ribose (sugar)-phosphate backbone (

Remo1al of primer
After replication of the desired re!ion, the *NA primer is remo"ed by DNA polymerase 3 "ia the process of nick translation. The remo"al of the *NA primer allows DNA li!ase to li!ate the DNA-DNA nick between the new fra!ment and the pre"ious strand. DNA polymerase 3 Z 333, alon! with many other enzymes are all re+uired for the hi!h fidelity, hi!h-processi"ity of DNA replication.

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