This strand displacing activity is very similar to that reported for bacterial Pol I. However, recent work has shown that FEN1 initially binds to the base of the flap causing a change in the substrate conformation that orients FEN1 in a manner that allows for precise cleavage Gloor et al. DNA ligase I cdc9 in S. It has been previously suggested that PCNA serves to recruit the core enzymes to the replication fork and functions to sequentially hand off the proteins to perform their enzymatic tasks during the maturation process Kao and Bambara Apparently the exonuclease regulates displacement Garg and Burgers b.

However, analysis of lagging-strand replication using purified S. Several displacement and cleavage reactions are required to remove the initiator primer. Okazaki fragment maturation. In some instances FEN1 transiently disengages from the replication complex. RPA-bound flaps are refractory to FEN1 cleavage, requiring the action of another nuclease for proper processing Bae et al.

Budd and Campbell identified this alternate nuclease, Dna2, in a genetic screen in S. These results imply that Dna2 works with FEN1 specifically to process long flaps. In addition, Dna2 was recently found to be complexed with FEN1 in human cell extracts, suggesting it as a physical and functional partner of FEN1 Balakrishnan et al.

Reconstitution experiments have shown that although the majority of the displaced flaps are processed by the short flap pathway, a minority require the long flap pathway.

Seo and colleagues originally proposed the Dna2 pathway as the primary means of Okazaki fragment processing Bae et al. Their proposal was likely influenced by genetic evidence in S. However, these observations can be misleading. Also, recent work from the Campbell laboratory showed that Dna2 interacts with Rad9, the damage checkpoint activator, participating in the double-strand break repair response. Why should this minor long flap pathway have evolved? This was likely owing to FEN1 binding the base of the flap and orienting itself in a position allowing for cleavage, before RPA binding could be inhibitory.

This observation appeared to remove the need for Dna2 in the processing pathway. Hence, why did this protein evolve to interact with the replication proteins? The answer to this question was provided by genetic studies in S. Importantly, in S. This result indicates that expression of Pif1 creates a need for Dna2. A reasonable interpretation is that Pif1 makes Dna2 necessary for long flap processing.

Because Pif1 complicates fragment processing, why has it evolved to interact with the lagging-strand synthesis machinery? An answer was also suggested by Seo and colleagues Ryu et al. Pif1 may fully displace some fragments. When the flap is created, it folds in a way that prevents cleavage by either FEN1 or Dna2. However, Pif1 can bind between the structure and the flap base and fully displace the fragment. The upstream fragment can then extend through the structured region.

This is effectively a third pathway of fragment processing Pike et al. Although the long flap pathway has evolved elegantly to process such flaps, evidence in vitro suggests that the short flap pathway is more commonly used, with each protein from the long flap pathway Dna2, Pif1, and RPA stimulating the function of FEN1, and so promoting the use of the short flap pathway Henry et al.

Cells are constantly being exposed to endogenous and exogenous stresses that cause oxidative damage to DNA bases. Base excision repair BER is the most commonly utilized means of dealing with these damaged bases. It proceeds via two pathways. However, if the dRP is oxidized, reduced, or otherwise altered, the lyase function does not work.

It is not clear whether the longest flaps would bind RPA with sufficient avidity to require Dna2; however, the recent report of Dna2 involvement in mitochondrial LP-BER suggests that some do Zheng et al. The overlap of proteins used for Okazaki fragment processing and LP-BER suggests that the two processes evolved from the same ancestral basic pathway. The overlap also suggests that regulatory mechanisms for one process will similarly influence the other. Most of the proteins involved in eukaryotic DNA replication experience various posttranslational modifications, regulating their enzymatic functions, subcellular localizations, or participation in a specific pathway.

This in turn decreases the processivity of polymerization. PCNA is modified by a diverse range of modifications such as acetylation, phosphorylation, and ubiquitination. FEN1 is posttranslationally modified by phosphorylation, methylation, and acetylation.

Acetylation greatly diminishes the cleavage function of FEN1 Hasan et al. Interestingly, a recent report shows that phosphorylation of FEN1 stimulates its sumoylation. This subsequently helps in the ubiquitination of the protein leading to degradation via the proteosome pathway, thereby regulating the levels of FEN1 in the cell Guo et al. Dna2 is also phosphorylated and acetylated.

Although most posttranslational modifications alter the function of individual proteins, recent evidence of the regulation of proteins by acetylation suggests a coherent hypothesis in which acetylation of replication proteins regulates the choice of a specific pathway for Okazaki fragment maturation. It has been known for some time that FEN1 can be acetylated by the histone acetyltransferase p Hasan et al.

We now know that this phenomenon makes more sense when viewed in the context of regulation of most lagging-strand replication proteins by acetylation. Suggestions of a more global regulation mechanism came from analyses of the effects of acetylation on the properties of other lagging-strand replication proteins. The p acetylase also reacts with Dna2, with multifold stimulation of nuclease, helicase, and ATPase activities Balakrishnan et al. Notably, alteration of the helicase activity allows Dna2 to drive the nuclease active site to the base of the original flap, and on some flaps even farther.

This results in a shifted cleavage distribution farther downstream. Preliminary results show that acetylation of the polymerase greatly augments its ability to perform strand displacement synthesis. This would normally be a genome stability problem because long flaps can form secondary structures that inhibit processing, and can recombine at ectopic sites.

However, the increased efficiency of Dna2 must prevent the flaps from actually achieving great length.

Instead, displacement will occur for a greater distance, and ultimate ligation will be delayed because the lowered activity of FEN1 will not be able to rapidly create nicks. The overall consequence of the modification is that, without actually making long flap intermediates, a longer patch of the downstream fragment would be removed and replaced.

Why would this be desirable? In higher eukaryotes, millions of to nt fragments are needed to make the lagging strand. This would only be desirable if it protects DNA that provides the organism with a selective advantage.

A reasonable interpretation is that lagging-strand replication is selectively regulated for fidelity. The p acetylase activates selected areas of chromatin for gene expression.

Although Okazaki fragment processing is one of the fundamental processes of life, it can be optimized in any particular organism for speed, fidelity, energy consumption, or some combination. Speed and energy consumption would appear to be most important in bacteria because they are competing with other rapidly growing cells. Moreover, occasional lethal mutations should not affect the success of the population.

The result is the evolution of a long fragment mechanism. Higher eukaryotes appear to have developed processing that is optimized for fidelity in active genes. This would appear best for survival through development, delay of cancers, and a long average life span. We especially thank Christopher Petrides and Athena Kantartzis for assistance with the figures.

Previous Section Next Section. Figure 1. Figure 2. Previous Section. Studies on DNA replication in the T4 bacteriophage in vitro system. Google Scholar. Okazaki fragment maturation in yeast. J Biol Chem : — RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes. Nature : — The changing view of Dna2. Cell Cycle 10 : — Eukaryotic lagging strand DNA replication employs a multi-pathway mechanism that protects genome integrity.

Long patch base excision repair proceeds via coordinated stimulation of the multienzyme DNA repair complex. Enzymes and reactions at the eukaryotic DNA replication fork. Proc Natl Acad Sci 92 : — A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function. Mol Cell Biol 17 : — A network of multi-tasking proteins at the DNA replication fork preserves genome stability.

PLoS Genet 1 : e CrossRef Medline Google Scholar. Mol Cell Biol 26 : — Polymerase dynamics at the eukaryotic DNA replication fork. Ribonuclease H: The enzymes in eukaryotes. FEBS J : — Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation.

Nat Struct Mol Biol 18 : — Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science : — Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication. The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin.

The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems. Partial functional deficiency of ED flap endonuclease-1 mutant in vitro and in vivo is due to defective cleavage of DNA substrates.

Crit Rev Biochem Mol Biol 40 : — How the cell deals with DNA nicks. Cell Cycle 4 : — Medline Web of Science Google Scholar. Flap endonuclease 1 mechanism analysis indicates flap base binding prior to threading. Sequential posttranslational modifications program FEN1 degradation during cell-cycle progression.

Mol Cell 47 : — Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p Mol Cell 7 : — Analysis of nucleotide pools in animal cells. Methods Cell Biol 7 : — Medline Google Scholar. Components of the secondary pathway stimulate the primary pathway of eukaryotic Okazaki fragment processing.

J Bacteriol : — Eukaryotic DNA polymerases. Annu Rev Biochem 71 : — Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro. Dna2 on the road to Okazaki fragment processing and genome stability in eukaryotes.

Crit Rev Biochem Mol Biol 45 : 71 — The protein components and mechanism of eukaryotic Okazaki fragment maturation. Crit Rev Biochem Mol Biol 38 : — Metabolic Molecules 2. Water 3. Protein 5. Enzymes 6. Cell Respiration 9. Photosynthesis 3: Genetics 1. Genes 2. Chromosomes 3. Meiosis 4. Inheritance 5. Genetic Modification 4: Ecology 1. Energy Flow 3. Carbon Cycling 4. Climate Change 5: Evolution 1. Evolution Evidence 2. Natural Selection 3. Classification 4. Cladistics 6: Human Physiology 1.

Digestion 2. The Blood System 3. Disease Defences 4. Gas Exchange 5. Homeostasis Higher Level 7: Nucleic Acids 1. DNA Structure 2.

Transcription 3. Translation 8: Metabolism 1.