DNAReplication: Mammalian

Abstract

Deoxyribonucleic acid (DNA) replication is an evolutionarily semi‐conserved process that involves a mechanism to unwind the parent DNA strand, the synthesis of identical daughter strands, and steps to terminate the sequence. Owing to the massive amount of genetic information needed to be copied error‐free, the replication of DNA within the mammalian chromosome is highly complex, requiring the simultaneous firing of multiple origins and processing of DNA at defined sites within the cells. This action requires the strict regulation and precise timing of numerous proteins, enzymes and substrates. These components critical to DNA replication will associate within these compartments in the cell to form replication factories during processing. Although many of the basic functions of DNA synthesis are well‐defined, there is still much yet to be understood regarding the machinery involved in the initiation, synthesis and termination of mammalian DNA replication. Key Concepts: Mammalian DNA replication occurs at specific regions within the cell. Proteins necessary for DNA replication come together to form ‘replication factories’ within the nucleus. The manner and timing of mammalian DNA replication is largely determined in G1 of the cell cycle. The selection and timing of each replication site is controlled by a combination of primary sequences, kinase activation, transcription, and local chromatin environment. The proteins thus far identified at the mammalian cell DNA replication fork include the MCM helicase complex, DNA pol α‐primase, PCNA, RFC, DNA pol δ, FEN1, RPA, DNA ligase I, topoisomerase I and II and RNAse H. Termination of mammalian DNA replication is thought to be dependent on physical barriers within the replication factories, as opposed to actual termination sequences within the DNA structure. In the event of replication fork stalling, repair pathways must be activated to halt cell cycle progression and repair the damage.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis

“…Because of the association of PCNA within the DNA replication apparatus of the cell and the co-migration of PCNA with other proteins having a role in the DNA replication process, it became important to establish whether this PCNA containing multiprotein complex could be the functional DNA synthesome that we had previously reported. It is also important to note that PCNA serves as a processivity factor, which is an essential component of the DNA synthetic process [14] carried-out by the in vitro SV40 DNA replication model system used in the analysis of the mammalian cell DNA synthetic process [28, 29]. The native gel showed two peaks of PCNA with a relative electrophoretic mobility between 800–1,000 kDa (Fig 3B, S3 Fig), at fractions 29 and 38, and a lower mol wt PCNA species which appeared at fraction 17.…”

“…Early studies described eukaryotic DNA replication as an ordered process involving the activity of many proteins that bind as needed for a particular step but dissociate from the complex when no longer needed [14, 15]. This simplified model was based on in vitro experiments with reconstituted proteins [32]; it was useful at identifying essential proteins and revealing steps in the DNA replication process.…”

“…Proteins previously identified as part of the human DNA synthesome include: DNA polymerases α, δ, and ɛ (Pol α, δ, and ɛ), DNA ligase I, topoisomerase I and II (topo I and II), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), replication protein A (RPA), DNA primase, flap endonuclease 1 (FEN1), poly(ADP-ribose) polymerase (PARP), and DNA methyltransferase (DNMT1) [2, 5–7, 9–13] to sequentially carry-out the biochemical reactions associated with the initiation, elongation and termination phases of the DNA replication process. Each of the identified protein components of the DNA synthesome have specific roles in mammalian DNA replication [14, 15]. In an effort to preserve the overall structure, while maintaining the activity of the replication protein complex, we utilized free flow electrophoresis (FFE) to purify the DNA synthesome.…”

Novel Peptide Therapeutic Approaches for Cancer Treatment

“…A common strategy of designing peptide-based therapy is to disrupt protein–protein interactions (PPIs) with known protein binding sites. PCNA is a homotrimer that encircles DNA and acts as a platform that binds and coordinate proteins at the replication fork [ 89 , 90 ]. It is thought to interact and regulate over a hundred proteins [ 91 ] with various functions in the cell.…”