Leigh A. Henricksen

Enzymes and Reactions at the Eukaryotic DNA Replication Fork

Many recent reviews of eukaryotic DNA replication have emphasized our current understanding of either the initiation and regulation of eukaryotic nuclear DNA replication (1, 2), the DNA polymerases and other proteins involved (3–11), or the entire range of knowledge of the replication process (12–17). Here, we will focus on recent findings concerning specific enzymatic reactions necessary for the growth of the eukaryotic replication fork.

Cleavage Specificity of Saccharomyces cerevisiae Flap Endonuclease 1 Suggests a Double-Flap Structure as the Cellular Substrate

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves substrates containing unannealed 5′-flaps during Okazaki fragment processing. Cleavage removes the flap at or near the point of annealing. The preferred substrate for archaeal FEN1 or the 5′-nuclease domains of bacterial DNA polymerases is a double-flap structure containing a 3′-tail on the upstream primer adjacent to the 5′-flap. We report that FEN1 inSaccharomyces cerevisiae (Rad27p) exhibits a similar specificity. Cleavage was most efficient when the upstream primer contained a 1-nucleotide 3′-tail as compared with the fully annealed upstream primer traditionally tested. The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. In contrast, a portion of the products from traditional flap substrates is not ligated. The 3′-OH of the upstream primer is not critical for double-flap recognition, because Rad27p is tolerant of modifications. However, the positioning of the 3′-nucleotide defines the site of cleavage. We have tested substrates having complementary tails that equilibrate to many structures by branch migration. FEN1 only cleaved those containing a 1-nucleotide 3′-tail. Equilibrating substrates containing 12-ribonucleotides at the end of the 5′-flap simulates the situation in vivo. Rad27p cleaves this substrate in the expected 1-nucleotide 3′-tail configuration. Overall, these results suggest that the double-flap substrate is formed and cleaved during eukaryotic DNA replication in vivo.

DNA Ligase I and Proliferating Cell Nuclear Antigen Form a Functional Complex

DNA ligase I is responsible for joining Okazaki fragments during DNA replication. An additional proposed role for DNA ligase I is sealing nicks generated during excision repair. Previous studies have shown that there is a physical interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA), another important component of DNA replication and repair. The results shown here indicate that human PCNA enhances the reaction rate of human DNA ligase I up to 5-fold. The stimulation is specific to DNA ligase I because T4 DNA ligase is not affected. Electrophoretic mobility shift assays indicate that PCNA improves the binding of DNA ligase I to the ligation site. Increasing the DNA ligase I concentration leads to a reduction in PCNA stimulation, consistent with PCNA-directed improvement of DNA ligase I binding to its DNA substrate. Two experiments show that PCNA is required to encircle duplex DNA to enhance DNA ligase I activity. Biotin-streptavidin conjugations at the ends of a linear substrate inhibit PCNA stimulation. PCNA cannot enhance ligation on a circular substrate without the addition of replication factor C, which is the protein responsible for loading PCNA onto duplex DNA. These results show that PCNA is responsible for the stable association of DNA ligase I to nicked duplex DNA.

Human DNA ligase I efficiently seals nicks in nucleosomes.

The access to DNA within nucleosomes is greatly restricted for most enzymes and trans‐acting factors that bind DNA. We report here that human DNA ligase I, which carries out the final step of Okazaki fragment processing and of many DNA repair pathways, can access DNA that is wrapped about the surface of a nucleosome in vitro and carry out its enzymatic function with high efficiency. In addition, we find that ligase activity is not affected by the binding of linker histone (H1) but is greatly influenced by the disposition of the core histone tail domains. These results suggest that the window of opportunity for human DNA ligase I may extend well beyond the first stages of chromatin reassembly after DNA replication or repair.

Identification of rad27 Mutations That Confer Differential Defects in Mutation Avoidance, Repeat Tract Instability, and Flap Cleavage

ABSTRACT In eukaryotes, the nuclease activity of Rad27p (Fen1p) is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5′ ends of Okazaki fragments. Genetic analysis of Saccharomyces cerevisiae also has identified a role for Rad27p in mutation avoidance. rad27Δ mutants display both a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result primarily from duplications of DNA sequences that are flanked by direct repeats. These observations suggested that Rad27p activities in DNA replication and repair could be altered by mutagenesis and specifically assayed. To test this idea, we analyzed two rad27alleles, rad27-G67S and rad27-G240D, that were identified in a screen for mutants that displayed repeat tract instability and mutator phenotypes. In chromosome stability assays,rad27-G67S strains displayed a higher frequency of repeat tract instabilities relative to CAN1 duplication events; in contrast, the rad27-G240D strains displayed the opposite phenotype. In biochemical assays, rad27-G67Sp displayed a weak exonuclease activity but significant single- and double-flap endonuclease activities. In contrast, rad27-G240Dp displayed a significant double-flap endonuclease activity but was devoid of exonuclease activity and showed only a weak single-flap endonuclease activity. Based on these observations, we hypothesize that the rad27-G67S mutant phenotypes resulted largely from specific defects in nuclease function that are important for degrading bubble intermediates, which can lead to DNA slippage events. Therad27-G240D mutant phenotypes were more difficult to reconcile to a specific biochemical defect, suggesting a structural role for Rad27p in DNA replication and repair. Since the mutants provide the means to relate nuclease functions in vitro to genetic characteristics in vivo, they are valuable tools for further analyses of the diverse biological roles of Rad27p.

Flap Endonuclease 1 Efficiently Cleaves Base Excision Repair and DNA Replication Intermediates Assembled into Nucleosomes

Flap Endonuclease 1 (FEN1) plays important roles both in DNA replication and in base excision repair (BER). However, in both processes FEN1 substrates are likely to be assembled into chromatin. In order to examine how FEN1 is able to work within chromatin, we prepared model nucleosome substrates containing FEN1-cleavable DNA flaps. We find that human FEN1 binds and cleaves such substrates with efficiencies similar to that displayed with naked DNA. Moreover, we demonstrate that both FEN1 and human DNA ligase I can operate successively on DNA within the same nucleosome. These results suggest that some BER steps may not require nucleosome remodeling in vivo and that FEN 1 activity during Okazaki fragment processing can occur on nucleosomal substrates.

VIP is a transcriptional target of Nurr1 in dopaminergic cells.

The orphan nuclear receptor Nurr1 is required for the development of the ventral mesencephalic dopaminergic neurons. These are the same neurons that are invariantly lost in patients with Parkinsons disease. Nurr1 mRNA expression is not confined to the developing midbrain, and yet Nurr1 appears to be essential for either the maturation of progenitors into fully post-mitotic dopaminergic neurons and/or once formed, their survival. The function of Nurr1 in the transactivation of gene(s) important for neuronal development and/or maintenance is uncharacterized. To characterize potential downstream target genes of Nurr1, we sought to identify mRNAs that are differentially affected by Nurr1 expression. Using a dopaminergic cell line in which Nurr1 content was tightly regulated, differential display analysis identified transcripts altered by Nurr1 expression, including the mRNA encoding vasoactive intestinal peptide (VIP). Herein, we demonstrate that Nurr1 regulates VIP mRNA and protein levels, and transactivates the VIP promoter through Nurr1-responsive cis elements. In addition, dopaminergic cells release and utilize VIP to mediate survival when challenged with paraquat. Nurr1 regulation of VIP is also demonstrated in vivo as loss of Nurr1 function results in diminished VIP mRNA levels within the developing midbrain.

CLEAVAGE OF SUBSTRATES WITH MISMATCHED NUCLEOTIDES BY FLAP ENDONUCLEASE-1 : IMPLICATIONS FOR MAMMALIAN OKAZAKI FRAGMENT PROCESSING

Flap endonuclease-1 (FEN1) is proposed to participate in removal of the initiator RNA of mammalian Okazaki fragments by two pathways. In one pathway, RNase HI removes most of the RNA, leaving a single ribonucleotide adjacent to the DNA. FEN1 removes this ribonucleotide exonucleolytically. In the other pathway, FEN1 removes the entire primer endonucleolytically after displacement of the 5′-end region of the Okazaki fragment. Cleavage would occur beyond the RNA, a short distance into the DNA. The initiator RNA and an adjacent short region of DNA are synthesized by DNA polymerase α/primase. Because the fidelity of DNA polymerase α is lower than that of the DNA polymerases that complete DNA extension, mismatches occur relatively frequently near the 5′-ends of Okazaki fragments. We have examined the ability of FEN1 to repair such errors. Results show that mismatched bases up to 15 nucleotides from the 5′-end of an annealed DNA strand change the pattern of FEN1 cleavage. Instead of removing terminal nucleotides sequentially, FEN1 appears to cleave a portion of the mismatched strand endonucleolytically. We propose that a mismatch destabilizes the helical structure over a nearby area. This allows FEN1 to cleave more efficiently, facilitating removal of the mismatch. If mismatches were not introduced during synthesis of the Okazaki fragment, helical disruption would not occur, nor would unnecessary degradation of the 5′-end of the fragment.