Top and side views of ahomotrimerof the humanPCNAsliding clamp (rainbow colored,N-terminus= blue,C-terminus= red) with double strandedDNAmodeled through the central pore (magenta).[1]
Cryo-EM structure of the DNA-bound PolD–PCNA processive complexStructural basis for DNA binding by the PolD–PCNA complex
ADNA clamp,also known as asliding clamp,is aprotein complexthat serves as aprocessivity-promoting factor inDNA replication.As a critical component of theDNA polymerase III holoenzyme,the clamp protein bindsDNA polymeraseand prevents thisenzymefrom dissociating from the templateDNAstrand. The clamp-polymeraseprotein–protein interactionsare stronger and more specific than the direct interactions between the polymerase and the template DNA strand; because one of therate-limiting stepsin the DNA synthesis reaction is the association of the polymerase with the DNA template, the presence of the sliding clamp dramatically increases the number ofnucleotidesthat the polymerase can add to the growing strand per association event. The presence of the DNA clamp can increase the rate of DNA synthesis up to 1,000-fold compared with a nonprocessive polymerase.[2]
The DNA clamp is anα+βprotein that assembles into a multimeric, six-domain ring structure that completely encircles the DNA double helix as the polymerase addsnucleotidesto the growing strand.[3]Each domain is in turn made of two β-α-β-β-β structural repeats.[4]The DNA clamp assembles on the DNA at thereplication forkand "slides" along the DNA with the advancing polymerase, aided by a layer ofwatermolecules in the central pore of the clamp between the DNA and the protein surface. Because of thetoroidalshape of the assembled multimer, the clamp cannot dissociate from the template strand without also dissociating intomonomers.
The DNA clamp fold is found inbacteria,archaea,eukaryotesand someviruses.In bacteria, the sliding clamp is ahomodimercomposed of two identical beta subunits ofDNA polymerase IIIand hence is referred to as the beta clamp. In archaea[5]and eukaryotes, it is a trimer composed of three molecules ofPCNA.TheT4 bacteriophagealso uses a sliding clamp, called gp45 that is a trimer similar in structure to PCNA but lacks sequence homology to either PCNA or the bacterial beta clamp.[3]
Thebeta clampis a specific DNA clamp and a subunit of theDNA polymerase IIIholoenzyme found in bacteria. Two beta subunits are assembled around the DNA by the gamma subunit and ATP hydrolysis; this assembly is called thepre-initiation complex.After assembly around the DNA, the beta subunits' affinity for the gamma subunit is replaced by an affinity for the Alpha and epsilon subunits, which together create the complete holoenzyme.[7][8][9]DNA polymerase III is the primary enzyme complex involved inprokaryoticDNA replication.
The gamma complex of DNA polymerase III, composed of γδδ'χψ subunits, catalyzesATPto chaperone two beta subunits to bind to DNA. Once bound to DNA, the beta subunits can freely slide along double stranded DNA. The beta subunits in turn bind the αε polymerase complex. The α subunit possessesDNA polymeraseactivity and the ε subunit is a 3’-5’exonuclease.[9]
The beta chain of bacterial DNA polymerase III is composed of three topologically equivalentdomains(N-terminal,central, andC-terminal). Two beta chain molecules are tightly associated to form a closed ring encircling duplex DNA.
The sliding clamp in eukaryotes is assembled from a specific subunit ofDNA polymerase deltacalled the proliferating cell nuclear antigen (PCNA). TheN-terminalandC-terminaldomains of PCNA are topologically identical. Three PCNA molecules are tightly associated to form a closed ring encircling duplex DNA.
The sequence of PCNA is well conserved between plants, animals and fungi, indicating a strong selective pressure for structure conservation, and suggesting that this type of DNA replication mechanism is conserved throughout eukaryotes.[12][13]In eukaryotes, a homologous, heterotrimeric "9-1-1 clamp" made up ofRAD9-RAD1-HUS1(911) is responsible for DNA damage checkpoint control.[14]This 9-1-1 clamp mounts onto DNA in the opposite direction.[15]
Archaea,probable evolutionary precursor of eukaryotes, also universally have at least one PCNA gene. This PCNA ring works withPolD,the single eukaryotic-like DNA polymerase in archaea responsible for multiple functions from replication to repair. Some unusual species have two or even three PCNA genes, forming heterotrimers or distinct specialized homotrimers.[16]Archaeons also share with eukaryotes the PIP (PCNA-interacting protein) motif, but a wider variety of such proteins performing different functions are found.[17]
PCNA is also appropriated by some viruses. The giant virus genusChlorovirus,with PBCV-1 as a representative, carries in its genome two PCNA genes (Q84513,O41056) and a eukaryotic-type DNA polymerase.[18]Members ofBaculoviridaealso encode a PCNA homolog (P11038).[19]
The viral gp45 sliding clamp subunit protein contains two domains. Each domain consists of two Alpha helices and two beta sheets – the fold is duplicated and has internal pseudo two-fold symmetry.[21]Three gp45 molecules are tightly associated to form a closed ring encircling duplex DNA.
Some members ofHerpesviridaeencode a protein that has a DNA clamp fold but does not associate into a ring clamp. The two-domain protein does, however, associate with the viral DNA polymerase and also acts to increase processivity.[22]As it does not form a ring, it does not need a clamp loader to be attached to DNA.[23]
DNA polymerase processivity factor (HSVUL42, Alphaherpesvirus)
Sliding clamps are loaded onto their associated DNA template strands by specialized proteins known as "sliding clamp loaders",which also disassemble the clamps after replication has completed. The binding sites for these initiator proteins overlap with the binding sites for the DNA polymerase, so the clamp cannot simultaneously associate with a clamp loader and with a polymerase. Thus the clamp will not be actively disassembled while the polymerase remains bound. DNA clamps also associate with other factors involved in DNA and genome homeostasis, such asnucleosomeassembly factors,Okazaki fragmentligases, andDNA repairproteins. All of these proteins also share a binding site on the DNA clamp that overlaps with the clamp loader site, ensuring that the clamp will not be removed while any enzyme is still working on the DNA. The activity of the clamp loader requiresATP hydrolysisto "close" the clamp around the DNA.
^Mizrahi V, Henrie RN, Marlier JF, Johnson KA, Benkovic SJ (July 1985). "Rate-limiting steps in the DNA polymerase I reaction pathway".Biochemistry.24(15): 4010–4018.doi:10.1021/bi00336a031.PMID3902078.
^Pan M, Kelman LM, Kelman Z (January 2011). "The archaeal PCNA proteins".Biochemical Society Transactions.39(1): 20–24.doi:10.1042/bst0390020.PMID21265741.
^Fu Y, Wang R, Liang A (June 2018). "Function analysis of Ac-PCNA and Sf-PCNA during the Autographa californica multiple nucleopolyhedrovirus infection process".Molecular and Cellular Biochemistry.443(1–2): 57–68.doi:10.1007/s11010-017-3210-y.PMID29075988.S2CID9507736.
^PDB:1CZD;Moarefi I, Jeruzalmi D, Turner J, O'Donnell M, Kuriyan J (March 2000). "Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage".Journal of Molecular Biology.296(5): 1215–1223.doi:10.1006/jmbi.1999.3511.PMID10698628.
Watson JD, Baker TA, Bell SP, Gann A, Levine M, Losick R (2004).Molecular Biology of the Gene.San Francisco: Pearson/Benjamin Cummings.ISBN978-0-8053-4635-0.
