Editing Primase

{{Short description|Class of enzymes}}
{{Infobox protein family|Name=Toprim domain|InterPro=IPR006171|Symbol=Toprim|Pfam=PF01751|Pfam_clan=Toprim-like|SCOP=2fcj}}
{{Infobox protein family|Name=Toprim catalytic core|Symbol=Toprim_N|InterPro=IPR013264|Pfam=PF08275|SCOP=1dd9}}
{{Infobox protein family|Name=AEP DNA primase, small subunit|Symbol=DNA_primase_S|Pfam=PF01896|InterPro=IPR002755|Pfam_clan=AEP|SCOP=1g71}}
{{Infobox protein family|Name=AEP DNA primase, large subunit|Symbol=DNA_primase_lrg|Pfam=PF04104|InterPro=IPR007238|Pfam_clan=CL0242|SCOP=1zt2}}

'''DNA primase''' is an [[enzyme]] involved in the [[DNA replication|replication of DNA]] and is a type of [[RNA polymerase]]. Primase catalyzes the synthesis of a short RNA (or DNA in some 
living organisms<ref name="Bocquier">{{cite journal | vauthors = Bocquier AA, Liu L, Cann IK, Komori K, Kohda D, Ishino Y | title = Archaeal primase: bridging the gap between RNA and DNA polymerases | journal = Current Biology | volume = 11 | issue = 6 | pages = 452–6 | date = March 2001 | pmid = 11301257 | doi = 10.1016/s0960-9822(01)00119-1 | doi-access = free }}</ref>) segment called a [[Primer (molecular biology)|primer]] complementary to a [[ssDNA]] (single-stranded DNA) template. After this elongation, the [[RNA]] piece is removed by a 5' to 3' [[exonuclease]] and refilled with DNA.

== Function ==
[[File:Asymmetry in the synthesis of leading and lagging strands.svg|thumb|left|Asymmetry in the synthesis of leading and lagging strands, with role of DNA primase shown]]
[[File:Steps in DNA synthesis.svg|thumb|left|Steps in DNA synthesis, with role of DNA primase shown]]

In [[bacteria]], primase binds to the [[DNA helicase]] forming a complex called the [[primosome]]. Primase is activated by the helicase where it then synthesizes a short RNA primer approximately 11 ±1 [[nucleotides]] long, to which new nucleotides can be added by DNA polymerase. [[Archaeal]] and [[eukaryote]] primases are [[heterodimeric]] proteins with one large regulatory and one minuscule catalytic subunit.<ref name="Crystal structure of the human prim">{{cite journal | vauthors = Baranovskiy AG, Zhang Y, Suwa Y, Babayeva ND, Gu J, Pavlov YI, Tahirov TH | title = Crystal structure of the human primase | journal = The Journal of Biological Chemistry | volume = 290 | issue = 9 | pages = 5635–46 | date = February 2015 | pmid = 25550159 | pmc = 4342476 | doi = 10.1074/jbc.M114.624742 | doi-access = free }}</ref>

The RNA segments are first synthesized by primase and then elongated by DNA polymerase.<ref name=griep>{{cite journal | vauthors = Griep MA | title = Primase structure and function | journal = Indian Journal of Biochemistry & Biophysics | volume = 32 | issue = 4 | pages = 171–8 | date = August 1995 | pmid = 8655184 }}</ref> Then the DNA polymerase forms a protein complex with two primase subunits to form the alpha DNA Polymerase primase complex. Primase is one of the most error prone and slow polymerases.<ref name="griep"/> Primases in organisms such as [[Escherichia coli|''E. coli'']] synthesize around 2000 to 3000 primers at the rate of one primer per second.<ref name=keck>{{cite journal | vauthors = Keck JL, Roche DD, Lynch AS, Berger JM | title = Structure of the RNA polymerase domain of E. coli primase | journal = Science | volume = 287 | issue = 5462 | pages = 2482–6 | date = March 2000 | pmid = 10741967 | doi = 10.1126/science.287.5462.2482 | bibcode = 2000Sci...287.2482K }}</ref>  Primase also acts as a halting mechanism to prevent the [[DNA replication#Leading strand|leading strand]] from outpacing the [[DNA replication#Lagging strand|lagging strand]] by halting the progression of the [[DNA replication#Replication fork|replication fork]].<ref name=lee>{{cite journal | vauthors = Lee JB, Hite RK, Hamdan SM, Xie XS, Richardson CC, van Oijen AM | title = DNA primase acts as a molecular brake in DNA replication | journal = Nature | volume = 439 | issue = 7076 | pages = 621–4 | date = February 2006 | pmid = 16452983 | doi = 10.1038/nature04317 | bibcode = 2006Natur.439..621L | s2cid = 3099842 | url = https://pure.rug.nl/ws/files/14490287/2006NatureLee.pdf }}</ref> The rate determining step in primase is when the first [[phosphodiester bond]] is formed between two molecules of RNA.<ref name =griep />

The replication mechanisms differ between different bacteria and [[virus]]es where the primase covalently link to [[helicase]] in viruses such as the [[T7 phage|T7 bacteriophage]].<ref name=lee /> In viruses such as the [[herpes simplex virus]] (HSV-1), primase can form complexes with helicase.<ref name=cav>{{cite journal | vauthors = Cavanaugh NA, Kuchta RD | title = Initiation of new DNA strands by the herpes simplex virus-1 primase-helicase complex and either herpes DNA polymerase or human DNA polymerase alpha | journal = The Journal of Biological Chemistry | volume = 284 | issue = 3 | pages = 1523–32 | date = January 2009 | pmid = 19028696 | pmc = 2615532 | doi = 10.1074/jbc.M805476200 | doi-access = free }}</ref> The primase-helicase complex is used to unwind dsDNA (double-stranded) and synthesizes the lagging strand using RNA primers<ref name=cav /> The majority of primers synthesized by primase are two to three nucleotides long.<ref name=cav />

== Types ==
There are two main types of primase: [[DnaG]] found in most bacteria, and the AEP (Archaeo-Eukaryote Primase) superfamily found in archaean and eukaryotic primases. While bacterial primases ([[DnaG]]-type) are composed of a single protein unit (a monomer) and synthesize RNA primers, AEP primases are usually composed of two different primase units (a heterodimer) and synthesize two-part primers with both RNA and DNA components.<ref>{{cite journal | vauthors = Keck JL, Berger JM | title = Primus inter pares (first among equals) | journal = Nature Structural Biology | volume = 8 | issue = 1 | pages = 2–4 | date = January 2001 | pmid = 11135655 | doi = 10.1038/82996 | s2cid = 17108681 }}</ref> While functionally similar, the two primase superfamilies evolved independently of each other.

=== DnaG ===
The crystal structure of primase in ''E. coli'' with a core containing the [[DnaG]] protein was determined in the year 2000.<ref name=keck />  The DnaG and primase complex is cashew shaped and contains three subdomains.<ref name=keck />  The central subdomain forms a [[toprim fold]] which is made of a mixture five [[beta sheet]]s and six [[Alpha helix|alpha helices]].<ref name=keck /><ref name=toprim>{{cite journal | vauthors = Aravind L, Leipe DD, Koonin EV | title = Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins | journal = Nucleic Acids Research | volume = 26 | issue = 18 | pages = 4205–13 | date = September 1998 | pmid = 9722641 | pmc = 147817 | doi = 10.1093/nar/26.18.4205 }}</ref>  The toprim fold is used for binding regulators and metals. The primase uses a [[Phosphotransferase|phosphotransfer]] domain for the transfer coordination of metals, which makes it distinct from other polymerases.<ref name=keck />  The side subunits contain a [[Amine|NH<sub>2</sub>]] and [[COOH-terminal]] made of alpha helixes and beta sheets.<ref name=keck /> The NH<sub>2</sub> terminal interacts with a [[zinc]] binding domain and COOH-terminal region which interacts with DnaB-ID.<ref name=keck />

The Toprim fold is also found in [[topoisomerase]] and mitochrondrial [[Twinkle (protein)|Twinkle]] primase/helicase.<ref name=toprim/> Some DnaG-like (bacteria-like; {{InterPro|IPR020607}}) primases have been found in archaeal genomes.<ref>{{cite journal | vauthors = Hou L, Klug G, Evguenieva-Hackenberg E | title = The archaeal DnaG protein needs Csl4 for binding to the exosome and enhances its interaction with adenine-rich RNAs | journal = RNA Biology | volume = 10 | issue = 3 | pages = 415–24 | date = March 2013 | pmid = 23324612 | pmc = 3672285 | doi = 10.4161/rna.23450 }}</ref>

=== AEP ===
Eukaryote and archaeal primases tend to be more similar to each other, in terms of structure and mechanism, than they are to bacterial primases.<ref>{{cite journal | vauthors = Iyer LM, Koonin EV, Leipe DD, Aravind L | title = Origin and evolution of the archaeo-eukaryotic primase superfamily and related palm-domain proteins: structural insights and new members | journal = Nucleic Acids Research | volume = 33 | issue = 12 | pages = 3875–96 | date = 2005 | pmid = 16027112 | pmc = 1176014 | doi = 10.1093/nar/gki702 }}</ref><ref name=":3" /> The archaea-eukaryotic primase (AEP) superfamily, which most eukaryal and archaeal primase catalytic subunits belong to, has recently been redefined as a primase-polymerase family in recognition of the many other roles played by enzymes in this family.<ref name="Guil2015" /> This classification also emphasizes the broad origins of AEP primases; the superfamily is now recognized as transitioning between RNA and DNA functions.<ref name=":5">{{cite journal | vauthors = Gill S, Krupovic M, Desnoues N, Béguin P, Sezonov G, Forterre P | title = A highly divergent archaeo-eukaryotic primase from the Thermococcus nautilus plasmid, pTN2 | journal = Nucleic Acids Research | volume = 42 | issue = 6 | pages = 3707–19 | date = April 2014 | pmid = 24445805 | pmc = 3973330 | doi = 10.1093/nar/gkt1385 }}</ref>

Archaeal and eukaryote primases are heterodimeric proteins with one large regulatory (human [[PRIM2]], p58) and one small catalytic subunit (human [[PRIM1]], p48/p49).<ref name="Crystal structure of the human prim"/> The large subunit contains a N-terminal 4Fe–4S cluster, split out in some archaea as PriX/PriCT.<ref name=KazlauskasJMB /> The large subunit is implicated in improving the activity and specificity of the small subunit. For example, removing the part corresponding to the large subunit in a fusion protein PolpTN2 results in a slower enzyme with reverse transcriptase activity.<ref name=":5"/>

== Multifunctional primases ==
[[File:Multifunctional primases figure.jpg|right|thumb|500px|'''Figure 1.''' Select multifunctional primases across three domains of life (eukaryota, archaea, and bacteria). The ability of a primase to perform a particular activity is indicated by a check mark. Adapted from.<ref name="Guil2015"/>]]

The AEP family of primase-polymerases has diverse features beyond making only primers. In addition to priming DNA during replication, AEP enzymes may have additional functions in the DNA replication process, such as [[Polymerase|polymerization]] of DNA or RNA, [[Terminal deoxynucleotidyl transferase|terminal transfer]], [[DNA repair#Translesion synthesis|translesion synthesis (TLS)]], [[Non-homologous end joining|non-homologous end joining (NHEJ)]],<ref name="Guil2015">{{cite journal | vauthors = Guilliam TA, Keen BA, Brissett NC, Doherty AJ | title = Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes | journal = Nucleic Acids Research | volume = 43 | issue = 14 | pages = 6651–64 | date = August 2015 | pmid = 26109351 | pmc = 4538821 | doi = 10.1093/nar/gkv625 }}</ref> and possibly in restarting stalled replication forks.<ref name=":1">{{cite journal | vauthors = Wan L, Lou J, Xia Y, Su B, Liu T, Cui J, Sun Y, Lou H, Huang J | title = hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity | journal = EMBO Reports | volume = 14 | issue = 12 | pages = 1104–12 | date = December 2013 | pmid = 24126761 | pmc = 3981091 | doi = 10.1038/embor.2013.159 }}</ref> Primases typically synthesize primers from [[ribonucleotide]]s (NTPs); however, primases with polymerase capabilities also have an affinity for [[deoxyribonucleotide]]s (dNTPs).<ref name=":2">{{cite journal | vauthors = García-Gómez S, Reyes A, Martínez-Jiménez MI, Chocrón ES, Mourón S, Terrados G, Powell C, Salido E, Méndez J, Holt IJ, Blanco L | title = PrimPol, an archaic primase/polymerase operating in human cells | language = English | journal = Molecular Cell | volume = 52 | issue = 4 | pages = 541–53 | date = November 2013 | pmid = 24207056 | pmc = 3899013 | doi = 10.1016/j.molcel.2013.09.025 }}</ref><ref name=":3">{{cite journal | vauthors = Lao-Sirieix SH, Bell SD | title = The heterodimeric primase of the hyperthermophilic archaeon Sulfolobus solfataricus possesses DNA and RNA primase, polymerase and 3'-terminal nucleotidyl transferase activities | journal = Journal of Molecular Biology | volume = 344 | issue = 5 | pages = 1251–63 | date = December 2004 | pmid = 15561142 | doi = 10.1016/j.jmb.2004.10.018 }}</ref> Primases with terminal transferase functionality are capable of adding nucleotides to the 3’ end of a DNA strand independently of a template. Other enzymes involved in DNA replication, such as helicases, may also exhibit primase activity.<ref name=":4">{{cite journal | vauthors = Sanchez-Berrondo J, Mesa P, Ibarra A, Martínez-Jiménez MI, Blanco L, Méndez J, Boskovic J, Montoya G | title = Molecular architecture of a multifunctional MCM complex | journal = Nucleic Acids Research | volume = 40 | issue = 3 | pages = 1366–80 | date = February 2012 | pmid = 21984415 | pmc = 3273815 | doi = 10.1093/nar/gkr831 }}</ref>

===In eukaryotes and archaea===

Human [[PrimPol]] (ccdc111<ref name=":2" />) serves both primase and polymerase functions, like many archaeal primases; exhibits terminal transferase activity in the presence of manganese; and plays a significant role in translesion synthesis<ref name=":6">{{cite journal | vauthors = Keen BA, Jozwiakowski SK, Bailey LJ, Bianchi J, Doherty AJ | title = Molecular dissection of the domain architecture and catalytic activities of human PrimPol | journal = Nucleic Acids Research | volume = 42 | issue = 9 | pages = 5830–45 | date = May 2014 | pmid = 24682820 | pmc = 4027207 | doi = 10.1093/nar/gku214 }}</ref> and in restarting stalled replication forks. PrimPol is actively recruited to damaged sites through its interaction with RPA, an adapter protein that facilitates DNA replication and repair.<ref name=":1" /> PrimPol has a zinc finger domain similar to that of some viral primases, which is essential for translesion synthesis and primase activity and may regulate primer length.<ref name=":6" /> Unlike most primases, PrimPol is uniquely capable of starting DNA chains with dNTPs.<ref name=":2" />

PriS, the archaeal primase small subunit, has a role in translesion synthesis (TLS) and can bypass common DNA lesions. Most archaea lack the specialized polymerases that perform TLS in eukaryotes and bacteria.<ref>{{cite journal | vauthors = Jozwiakowski SK, Borazjani Gholami F, Doherty AJ | title = Archaeal replicative primases can perform translesion DNA synthesis | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 112 | issue = 7 | pages = E633-8 | date = February 2015 | pmid = 25646444 | pmc = 4343091 | doi = 10.1073/pnas.1412982112 | bibcode = 2015PNAS..112E.633J | doi-access = free }}</ref> PriS alone preferentially synthesizes strings of DNA; but in combination with PriL, the large subunit, RNA polymerase activity is increased.<ref>{{cite journal | vauthors = Barry ER, Bell SD | title = DNA replication in the archaea | journal = Microbiology and Molecular Biology Reviews | volume = 70 | issue = 4 | pages = 876–87 | date = December 2006 | pmid = 17158702 | pmc = 1698513 | doi = 10.1128/MMBR.00029-06 }}</ref>

In ''Sulfolobus solfataricus'', the primase heterodimer PriSL can act as a primase, polymerase, and terminal transferase. PriSL is thought to initiate primer synthesis with NTPs and then switch to dNTPs. The enzyme can polymerize RNA or DNA chains, with DNA products reaching as long as 7000 nucleotides (7 kb). It is suggested that this dual functionality may be a common feature of archaeal primases.<ref name=":3" />

===In bacteria===

AEP multifunctional primases also appear in bacteria and phages that infect them. They can display novel domain organizations with domains that bring even more functions beyond polymerization.<ref name=KazlauskasJMB />

Bacterial LigD ({{uniprot|A0R3R7}}) is primarily involved in the NHEJ pathway. It has an AEP superfamily polymerase/primase domain, a 3'-phosphoesterase domain, and a ligase domain. It is also capable of primase, DNA and RNA polymerase, and terminal transferase activity. DNA polymerization activity can produce chains over 7000 nucleotides (7 kb) in length, while RNA polymerization produces chains up to 1 kb long.<ref>{{cite journal | vauthors = Lao-Sirieix SH, Pellegrini L, Bell SD | title = The promiscuous primase | language = English | journal = Trends in Genetics | volume = 21 | issue = 10 | pages = 568–72 | date = October 2005 | pmid = 16095750 | doi = 10.1016/j.tig.2005.07.010 }}</ref>

=== In viruses and plasmids ===
AEP enzymes are widespread, and can be found encoded in mobile genetic elements including virus/phages and plasmids. They either use them as a sole replication protein or in combination with other replication-associated proteins, such as helicases and, less frequently, DNA polymerases.<ref name="KazlauskasNAR">{{cite journal | vauthors = Kazlauskas D, Krupovic M, Venclovas Č | title = The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes | journal = Nucleic Acids Research | volume = 44 | issue = 10 | pages = 4551–64 | date = June 2016 | pmid = 27112572 | pmc = 4889955 | doi = 10.1093/nar/gkw322 }}</ref> Whereas the presence of AEP in eukaryotic and archaeal viruses is expected in that they mirror their hosts,<ref name="KazlauskasNAR"/> bacterial viruses and plasmids also as frequently encode AEP-superfamily enzymes as they do DnaG-family primases.<ref name="KazlauskasJMB">{{cite journal | vauthors = Kazlauskas D, Sezonov G, Charpin N, Venclovas Č, Forterre P, Krupovic M | title = Novel Families of Archaeo-Eukaryotic Primases Associated with Mobile Genetic Elements of Bacteria and Archaea | journal = Journal of Molecular Biology | volume = 430 | issue = 5 | pages = 737–750 | date = March 2018 | pmid = 29198957 | pmc = 5862659 | doi = 10.1016/j.jmb.2017.11.014 }}</ref> A great diversity of AEP families has been uncovered in various bacterial plasmids by [[comparative genomics]] surveys.<ref name=KazlauskasJMB /> Their evolutionary history is currently unknown, as these found in bacteria and bacteriophages appear too different from their archaeo-eukaryotic homologs for a recent [[horizontal gene transfer]].<ref name="KazlauskasNAR"/>

[[Minichromosome maintenance|MCM-like helicase]] in ''Bacillus cereus'' strain ATCC 14579 (BcMCM; {{uniprot|Q81EV1}}) is an SF6 helicase fused with an AEP primase. The enzyme has both primase and polymerase functions in addition to helicase function. The gene coding for it is found in a prophage.<ref name=":4" /> It bears homology to ORF904 of plasmid pRN1 from ''Sulfolobus islandicus'', which has an AEP PrimPol domain.<ref>{{cite journal | vauthors = Lipps G, Weinzierl AO, von Scheven G, Buchen C, Cramer P | title = Structure of a bifunctional DNA primase-polymerase | journal = Nature Structural & Molecular Biology | volume = 11 | issue = 2 | pages = 157–62 | date = February 2004 | pmid = 14730355 | doi = 10.1038/nsmb723 | s2cid = 25123984 }}</ref> Vaccinia virus D5 and HSV Primase are examples of AEP-helicase fusion as well.<ref name="Guil2015"/><ref name=cav/>

PolpTN2 is an Archaeal primase found in the TN2 plasmid. A fusion of domains homologous to PriS and PriL, it exhibits both primase and DNA polymerase activity, as well as terminal transferase function. Unlike most primases, PolpTN2 forms primers composed exclusively of dNTPs.<ref name=":5" /> Unexpectedly, when the PriL-like domain was truncated, PolpTN2 could also synthesize DNA on the RNA template, i.e., acted as an RNA-dependent DNA polymerase (reverse transcriptase).<ref name=":5" />

Even DnaG primases can have extra functions, if given the right domains. The [[T7 DNA helicase|T7 phage gp4]] is a DnaG primase-helicase fusion, and performs both functions in replication.<ref name=lee />

== References ==
{{Reflist}}

== External links ==
*[http://digitalcommons.unl.edu/chemfacpub/2/ Overview article on primase structure and function (1995)]
*{{MeshName|DNA+Primase}}
*[http://proteopedia.org/wiki/index.php/2haj Proteopedia: Helicase-binding domain of Escherichia coli primase]
*[http://proteopedia.org/wiki/index.php/2r6c  Proteopedia: Complex between the DnaB helicase and the DnaG primase]

{{DNA replication}}
{{Kinases}}
{{Enzymes}}
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[[Category:EC 2.7.7]]
[[Category:DNA replication]]