A proposal: Evolution of PCNA's role as a marker of newly replicated DNA

Abstract

Processivity clamps that hold DNA polymerases to DNA for processivity were the first proteins known to encircle the DNA duplex. At the time, polymerase processivity was thought to be the only function of ring shaped processivity clamps. But studies from many laboratories have identified numerous proteins that bind and function with sliding clamps. Among these processes are mismatch repair and nucleosome assembly. Interestingly, there exist polymerases that are highly processive and do not require clamps. Hence, DNA polymerase processivity does not intrinsically require that sliding clamps evolved for this purpose. We propose that polymerases evolved to require clamps as a way of ensuring that clamps are deposited on newly replicated DNA. These clamps are then used on the newly replicated daughter strands, for processes important to genomic integrity, such as mismatch repair and the assembly of nucleosomes to maintain epigenetic states of replicating cells during development.

Keywords: DNA replication; PCNA; β-Clamp.

Figure. 1. Proposed use of DNA in LUCA

The ribosome, genetic code, tRNAs, and the DNA dependent RNA polymerase of RNA transcription are homologous in all three cellular domains of life, bacteria, archaea and eukaryotes. Thus, in this proposal, the translational and transcriptional pathways we re well developed in LUCA. The presence of Ribonucleotide Reductase, Recombinase and Thymidylate Synthase indicate that DNA was present, but the lack of homologous replication proteins suggest the DNA was not the genomic material. Proposed here, the DNA se rved the purpose of substrate for DNA recombination and transcription of mRNA and genomic RNA. Clamps and clamp loaders were present in LUCA, but their function is uncertain.

Figure. 2. Replisomes of bacteria and eukaryotes

A) The bacterial replisome is organized by the clamp loader, which contains a tau subunit homotrimer with extensions that bind three C-family DNA polymerases and connect to the helicase. The ho mohexameric helicase encircles the lagging strand. Primase is a single subunit based on the Toprim fold and acts to prime synthesis. DNA loops form during Okazaki fragment synthesis as a consequence of the connection between the leading and lagging strand polymerases via the clamp loader. B) The eukaryotic replisome is organized by the 11-subunit CMG helicase (composed of Cdc45, the Mcm2-7 hexamer that encircles the leading strand and the 4-subunit GINS heterotetramer. GINS binds to Pol ε, a B-family polymerase dedicated to the leading strand. The 4-subunit Pol α – Primase interacts with CMG through the Ctf4 homotrimer which also binds a GINS subunit of CMG. Pol α also contains a B-family polymerase that extends RNA primers to form hybrid RNA-DNA primers. Primers are further extended into Okazaki fragments by Pol δ (B-family polymerase) that functions with the PCNA clamp. Direct connections of Pol δ and the RFC clamp loader to other replisome components are currently unknown, unknown, and thus the lagging strand DNA may not form loops. The bacterial SSB tetramer and eukaryotic RPA ssDNA-binding proteins protect lagging strand ssDNA from nucleases and are not shown for clarity.

Figure. 3. Architecture of sliding clamps

A) Front view of the E. coli beta [pdb id:2POL] and yeast PCNA [pdb id:1PLQ] sliding clamps. Both rings are composed of six domains with the same chain folding topology. The six domains are arranged on a dimer of E. coli beta, and a trimer of eukaryotic PCNA. B) Side view of the clamps, showing the inherent asymmetry of the C- and N-terminal faces. Most clamp interactive proteins bind the C-face.

Figure. 4. Overview of clamp loader mechanism

Clamp loaders are circular heteropentamers with ATP sites situated at subunit interfaces. Three domains in each subunit include the N-terminal AAA+ domains and a C-terminal oligomerization domain referred to as a collar. ATP binding enables binding to the C-face of the clamp, opening it at one interface. Primed DNA fits into a central chamber, accessed through a gap between two clamp loader subunits and the open clamp interface. The DNA brings the subunits and clamp into a right-hand spiral, triggering ATP hydrolysis that ejects the clamp loader, enabling the clamp to close around DNA. Polymerase, or other proteins, can then interact with the clamp.

Figure. 5. PCNA marks newly replicated daughter strands for MMR and nucleosome assembly

Eukaryotic factors are illustrated. A) PCNA clamps are deposited on each Okazaki fragment by virtue of Pol δ dependency on PCNA for function. Upon completing an Okazaki fragment, Pol δ ejects from its PCNA clamp and binds a new clamp at the next RNA primer, leaving PCNA on the newly replicated DNA. B) Proposed process of populating the leading strand with PCNA. Pol ε-CMG does not absolutely require PCNA, but Pol ε has weak affinity for DNA and likely comes on and off the 3’ terminus during leading strand extension, staying bound to the fork through connection to CMG. This provides RFC access to the leading strand for PCNA clamp loading. PCNA marks newly replicated daughter strands for MMR and nucleosome assembly.