DNA clamp

A DNA clamp, also known as a sliding clamp, is a protein complex that serves as a processivity-promoting factor in DNA replication. As a critical component of the DNA polymerase III holoenzyme, the clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand. The clamp-polymerase protein–protein interactions are stronger and more specific than the direct interactions between the polymerase and the template DNA strand; because one of the rate-limiting steps in the DNA synthesis reaction is the association of the polymerase with the DNA template, the presence of the sliding clamp dramatically increases the number of nucleotides that 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]

Top and side views of a homotrimer of the human PCNA sliding clamp (rainbow colored, N-terminus = blue, C-terminus = red) with double stranded DNA modeled through the central pore (magenta).[1]
Cryo-EM structure of the DNA-bound PolD–PCNA processive complex
Structural basis for DNA binding by the PolD–PCNA complex

Structure

The DNA clamp is an α+β protein that assembles into a multimeric, six-domain ring structure that completely encircles the DNA double helix as the polymerase adds nucleotides to 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 the replication fork and "slides" along the DNA with the advancing polymerase, aided by a layer of water molecules in the central pore of the clamp between the DNA and the protein surface. Because of the toroidal shape of the assembled multimer, the clamp cannot dissociate from the template strand without also dissociating into monomers.

The DNA clamp fold is found in bacteria, archaea, eukaryotes and some viruses. In bacteria, the sliding clamp is a homodimer composed of two identical beta subunits of DNA polymerase III and hence is referred to as the beta clamp. In archaea[5] and eukaryotes, it is a trimer composed of three molecules of PCNA. The T4 bacteriophage also 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]

TaxonSliding clamp proteinMultimer stateAssociated polymerase
Bacteriabeta subunit of pol IIIdimerDNA polymerase III
Archaeaarchaeal PCNAtrimerPolD
EukaryotePCNAtrimerDNA polymerase delta
CaudoviralesIPR004190trimerPhage polymerase (e.g. T4)
Herpesviridaenon-clamp processivity factormonomerVirus-encoded polymerase

Bacterial
DNA polymerase III subunit beta
Crystallographic structure of the dimeric DNA polymerase beta subunit from E. coli.[6]
Identifiers
OrganismEscherichia coli
SymboldnaN
Entrez948218
PDB1MMI
RefSeq (Prot)NP_418156
UniProtP0A988
Other data
EC number2.7.7.7
ChromosomeMG1655: 3.88 - 3.88 Mb
Search for
StructuresSwiss-model
DomainsInterPro

The beta clamp is a specific DNA clamp and a subunit of the DNA polymerase III holoenzyme found in bacteria. Two beta subunits are assembled around the DNA by the gamma subunit and ATP hydrolysis; this assembly is called the pre-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 in prokaryotic DNA replication.

The gamma complex of DNA polymerase III, composed of γδδ'χψ subunits, catalyzes ATP to 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 possesses DNA polymerase activity and the ε subunit is a 3’-5’ exonuclease.[9]

The beta chain of bacterial DNA polymerase III is composed of three topologically equivalent domains (N-terminal, central, and C-terminal). Two beta chain molecules are tightly associated to form a closed ring encircling duplex DNA.

DNA polymerase III, beta chain (whole protein)
Identifiers
SymbolDNA_polIII_beta
InterProIPR001001
SMARTSM00480
SCOP22pol / SCOPe / SUPFAM
Available protein structures:
Pfam   
PDB1jqj​, 1jql​, 1mmi​, 1ok7​, 1unn​, 1vpk​, 2pol​, 3bep​, 3d1e​, 3d1f​, 3d1g​
DNA polymerase III, beta chain,
N-terminal
Identifiers
SymbolDNA_pol3_beta
PfamPF00712
InterProIPR022634
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
DNA polymerase III, beta chain,
central
Identifiers
SymbolDNA_pol3_beta_2
PfamPF02767
InterProIPR022637
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
DNA polymerase III, beta chain,
C-terminal
Identifiers
SymbolDNA_pol3_beta_3
PfamPF02768
InterProIPR022635
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

As a drug target

Certain NSAIDs (carprofen, bromfenac, and vedaprofen) exhibit some suppression of bacterial DNA replication by inhibiting bacterial DNA clamp.[10]

Eukaryotic and archaeal
proliferating cell nuclear antigen
The assembled human DNA clamp, a trimer of the human protein PCNA.[11]
Identifiers
SymbolPCNA
NCBI gene5111
HGNC8729
OMIM176740
PDB1axc
RefSeqNM_002592
UniProtP12004
Other data
EC number2.7.7.7
LocusChr. 20 pter-p12
Search for
StructuresSwiss-model
DomainsInterPro

The sliding clamp in eukaryotes is assembled from a specific subunit of DNA polymerase delta called the proliferating cell nuclear antigen (PCNA). The N-terminal and C-terminal domains 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 of RAD9-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 with PolD, 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 genus Chlorovirus, with PBCV-1 as a representative, carries in its genome two PCNA genes (Q84513, O41056) and a eukaryotic-type DNA polymerase.[18] Members of Baculoviridae also encode a PCNA homolog (P11038).[19]

Proliferating cell nuclear antigen, N-terminal domain
Identifiers
SymbolPCNA_N
PfamPF00705
InterProIPR000730
PROSITEPDOC00265
SCOP21plq / SCOPe / SUPFAM
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
PDB1axc​C:1–125 1ge8​A:3–92 1isq​A:3–92 1iz4​A:3–92 1iz5​A:3–92 1plq​ :1–125 1plr​ :1–125 1rwz​A:1–114 1rxm​A:1–114 1rxz​A:1–114 1u76​C:1–125 1u7b​A:1–125 1ud9​C:11–100 1ul1​A:1–125 1vyj​G:1–125 1vym​C:1–125 1w60​B:1–125
Proliferating cell nuclear antigen, C-terminal domain
Identifiers
SymbolPCNA_C
PfamPF02747
InterProIPR000730
PROSITEPDOC00265
SCOP21plq / SCOPe / SUPFAM
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
PDB1axc​C:127–254 1ge8​A:203–246 1isq​A:203–246 1iz4​A:203–246 1iz5​A:203–246 1plq​ :127–254 1plr​ :127–254 1rwz​A:121–241 1rxm​A:121–241 1rxz​A:121–241 1u76​C:127–254 1u7b​A:127–254 1ud9​C:200–243 1ul1​A:127–254 1vyj​G:127–254 1vym​C:127–254 1w60​B:127–254

Caudoviral
DNA polymerase accessory protein 45
Crystallographic structure of the trimeric gp45 sliding clamp from bacteriophage T4.[20]
Identifiers
OrganismEnterobacteria phage T4
Symbolgp45
Entrez1258821
PDB1CZD
RefSeq (Prot)NP_049666
UniProtP04525
Other data
EC number2.7.7.7
Chromosome1: 0.03 - 0.03 Mb
Search for
StructuresSwiss-model
DomainsInterPro

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.

Gp45 sliding clamp, N-terminal
Identifiers
SymbolDNA_pol_proc_fac
PfamPF02916
InterProIPR004190
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
PDB1b77​ 1b8h​ 1czd​
Gp45 sliding clamp, C-terminal
Identifiers
SymbolGp45_slide_clamp_C
PfamPF09116
InterProIPR015200
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
PDB1b77​ 1b8h​ 1czd​

Herpesviral

Some members of Herpesviridae encode 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 (HSV UL42, Alphaherpesvirus)
Identifiers
SymbolHerpes_UL42
PfamPF02282
InterProIPR003202
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
Herpesvirus polymerase accessory protein (Betaherpesvirus)
Identifiers
SymbolHerpes_PAP
PfamPF03325
InterProIPR004997
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
Herpes DNA replication accessory factor (Gammaherpesvirus)
Identifiers
SymbolHerpes_DNAp_acc
PfamPF04929
InterProIPR007013
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

Assembly

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 as nucleosome assembly factors, Okazaki fragment ligases, and DNA repair proteins. 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 requires ATP hydrolysis to "close" the clamp around the DNA.

References
  1. PDB: 1W60​; Kontopidis G, Wu SY, Zheleva DI, Taylor P, McInnes C, Lane DP, et al. (February 2005). "Structural and biochemical studies of human proliferating cell nuclear antigen complexes provide a rationale for cyclin association and inhibitor design". Proceedings of the National Academy of Sciences of the United States of America. 102 (6): 1871–1876. doi:10.1073/pnas.0406540102. PMC 548533. PMID 15681588.
  2. 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. PMID 3902078.
  3. Bruck I, O'Donnell M (2001). "The ring-type polymerase sliding clamp family". Genome Biology. 2 (1): REVIEWS3001. doi:10.1186/gb-2001-2-1-reviews3001. PMC 150441. PMID 11178284.
  4. Neuwald AF, Poleksic A (September 2000). "PSI-BLAST searches using hidden markov models of structural repeats: prediction of an unusual sliding DNA clamp and of beta-propellers in UV-damaged DNA-binding protein". Nucleic Acids Research. 28 (18): 3570–3580. doi:10.1093/nar/28.18.3570. PMC 110734. PMID 10982878.
  5. Matsumiya S, Ishino Y, Morikawa K (January 2001). "Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen from Pyrococcus furiosus". Protein Science. 10 (1): 17–23. doi:10.1110/ps.36401. PMC 2249843. PMID 11266590.
  6. PDB: 1MMI​; Oakley AJ, Prosselkov P, Wijffels G, Beck JL, Wilce MC, Dixon NE (July 2003). "Flexibility revealed by the 1.85 A crystal structure of the beta sliding-clamp subunit of Escherichia coli DNA polymerase III" (PDF). Acta Crystallographica. Section D, Biological Crystallography. 59 (Pt 7): 1192–1199. doi:10.1107/S0907444903009958. PMID 12832762.
  7. Lewin B (1997). Genes VI. Oxford [Oxfordshire]: Oxford University Press. pp. 484–7. ISBN 978-0-19-857779-9.
  8. Lehninger AL (1975). Biochemistry: The Molecular Basis of Cell Structure and Function. New York: Worth Publishers. pp. 894. ISBN 978-0-87901-047-8.
  9. Stukenberg PT, Studwell-Vaughan PS, O'Donnell M (June 1991). "Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme". The Journal of Biological Chemistry. 266 (17): 11328–11334. doi:10.1016/S0021-9258(18)99166-0. PMID 2040637.
  10. Yin Z, Wang Y, Whittell LR, Jergic S, Liu M, Harry E, et al. (April 2014). "DNA replication is the target for the antibacterial effects of nonsteroidal anti-inflammatory drugs". Chemistry & Biology. 21 (4): 481–487. doi:10.1016/j.chembiol.2014.02.009. PMID 24631121.
  11. PDB: 1AXC​; Gulbis JM, Kelman Z, Hurwitz J, O'Donnell M, Kuriyan J (October 1996). "Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA". Cell. 87 (2): 297–306. doi:10.1016/S0092-8674(00)81347-1. PMID 8861913. S2CID 17461501.
  12. Suzuka I, Hata S, Matsuoka M, Kosugi S, Hashimoto J (January 1991). "Highly conserved structure of proliferating cell nuclear antigen (DNA polymerase delta auxiliary protein) gene in plants". European Journal of Biochemistry. 195 (2): 571–575. doi:10.1111/j.1432-1033.1991.tb15739.x. PMID 1671766.
  13. Marshall AC, Kroker AJ, Murray LA, Gronthos K, Rajapaksha H, Wegener KL, Bruning JB (March 2017). "Structure of the sliding clamp from the fungal pathogen Aspergillus fumigatus (AfumPCNA) and interactions with Human p21". The FEBS Journal. 284 (6): 985–1002. doi:10.1111/febs.14035. PMID 28165677.
  14. Majka J, Burgers PM (2004). "The PCNA-RFC families of DNA clamps and clamp loaders". Progress in Nucleic Acid Research and Molecular Biology. 78: 227–260. doi:10.1016/S0079-6603(04)78006-X. ISBN 9780125400787. PMID 15210332.
  15. Zheng F, Georgescu RE, Yao NY, O'Donnell ME, Li H (April 2022). "DNA is loaded through the 9-1-1 DNA checkpoint clamp in the opposite direction of the PCNA clamp". Nature Structural & Molecular Biology. 29 (4): 376–385. doi:10.1038/s41594-022-00742-6. PMC 9010301. PMID 35314830.
  16. Pan M, Kelman LM, Kelman Z (January 2011). "The archaeal PCNA proteins". Biochemical Society Transactions. 39 (1): 20–24. doi:10.1042/bst0390020. PMID 21265741.
  17. MacNeill SA (August 2016). "PCNA-binding proteins in the archaea: novel functionality beyond the conserved core". Current Genetics. 62 (3): 527–532. doi:10.1007/s00294-016-0577-3. PMC 4929162. PMID 26886233.
  18. Van Etten JL, Agarkova IV, Dunigan DD (December 2019). "Chloroviruses". Viruses. 12 (1): 20. doi:10.3390/v12010020. PMC 7019647. PMID 31878033.
  19. 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. PMID 29075988. S2CID 9507736.
  20. 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. PMID 10698628.
  21. Shamoo Y, Steitz TA (October 1999). "Building a replisome from interacting pieces: sliding clamp complexed to a peptide from DNA polymerase and a polymerase editing complex". Cell. 99 (2): 155–166. doi:10.1016/S0092-8674(00)81647-5. PMID 10535734. S2CID 18103622.
  22. Zuccola HJ, Filman DJ, Coen DM, Hogle JM (February 2000). "The crystal structure of an unusual processivity factor, herpes simplex virus UL42, bound to the C terminus of its cognate polymerase". Molecular Cell. 5 (2): 267–278. doi:10.1016/S1097-2765(00)80422-0. PMID 10882068.
  23. Neuwald AF, Poleksic A (September 2000). "PSI-BLAST searches using hidden markov models of structural repeats: prediction of an unusual sliding DNA clamp and of beta-propellers in UV-damaged DNA-binding protein". Nucleic Acids Research. 28 (18): 3570–3580. doi:10.1093/nar/28.18.3570. PMC 110734. PMID 10982878.

Further reading
  • Clamping down on pathogenic bacteria – how to shut down a key DNA polymerase complex. Quips at PDBe
  • Watson JD, Baker TA, Bell SP, Gann A, Levine M, Losick R (2004). Molecular Biology of the Gene. San Francisco: Pearson/Benjamin Cummings. ISBN 978-0-8053-4635-0.
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