First discovered as a structure-specific endonuclease that evolved to cut at the base of single-stranded flaps, flap endonuclease (FEN1) is now recognized as a central component of cellular DNA metabolism. Substrate specificity allows FEN1 to process intermediates of Okazaki fragment maturation, long-patch base excision repair, telomere maintenance, and stalled replication fork rescue. For Okazaki fragments, the RNA primer is displaced into a 5′ flap and then cleaved off. FEN1 binds to the flap base and then threads the 5′ end of the flap through its helical arch and active site to create a configuration for cleavage. The threading requirement prevents this active nuclease from cutting the single-stranded template between Okazaki fragments. FEN1 efficiency and specificity are critical to the maintenance of genome fidelity. Overall, recent advances in our knowledge of FEN1 suggest that it was an ancient protein that has been fine-tuned over eons to coordinate many essential DNA transactions.

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Cellular DNA replication requires efficient copying of the double-stranded chromosomal DNA. The leading strand is elongated continuously in the direction of fork opening, whereas the lagging strand is made discontinuously in the opposite direction. The lagging strand needs to be processed to form a functional DNA segment. Genetic analyses and reconstitution experiments identified proteins and multiple pathways responsible for maturation of the lagging strand. In both prokaryotes and eukaryotes the lagging-strand fragments are initiated by RNA primers, which are removed by a joining mechanism involving strand displacement of the primer into a flap, flap removal, and then ligation. Although the prokaryotic fragments are 1200 nucleotides long, the eukaryotic fragments are much shorter, with lengths determined by nucleosome periodicity. The prokaryotic joining mechanism is simple and efficient. The eukaryotic maturation mechanism involves many enzymes, possibly three pathways, and regulation that can shift from high efficiency to high fidelity.

Summary Trinucleotide repeat (TNR) expansions are the underlying cause of more than forty neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington’s disease. Although genetic evidence has attributed the cause of these diseases to errors in DNA replication and/or repair, clear molecular mechanisms have not been described. We have focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. We further provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA Ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging strand DNA replication.

The pattern of organization of RNA polymerase II (RNAPII) in wild-type and mutant cs1085 SV40 chromosomes isolated between 30 min and 48 h post-infection was determined using a combination of chromatin immunoprecipitation (ChIP) techniques. During the course of a wild-type infection, we observed a slow but significant decline in the relative occupancy of RNAPII at the early region and a corresponding increase in occupation in the late region. In the promoter, occupancy began high, decreased to a minimum at 8 h post-infection, and then increased to a high level by 48 h post-infection. In the mutant cs1085, which does not down-regulate early transcription, we observed high occupancy of the early region and the promoter throughout the infection. The changing organization of RNAPII on the wild-type SV40 but not the mutant cs1085 genome appears to be a result of the switch from early to late transcription.

FEN1cleaves 5 flaps at their base to create a nicked product for ligation. FEN1 has been reported to enter the flap from the 5-end and track to the base. Current binding analyses support a very different mechanism of interaction with the flap substrate. Measurements of FEN1 binding to a flap substrate show that the nuclease binds with similar high affinity to the base of a long flap even when the 5-end is blocked with biotin/streptavidin. However, FEN1 bound to a blocked flap is more sensitive to sequestration by a competing substrate. These results are consistent with a substrate interaction mechanism in which FEN1 first binds the flap base and then threads the flap through an opening in the protein from the 5-end to the base for cleavage. Significantly, when the unblocked flap length is reduced from five to two nucleotides, FEN1 can be sequestered from the substrate to a similar extent as a blocked, long flap substrate. Apparently, interactions related to threading occur only when the flap is greater than two to four nucleotides long, implying that short flaps are cleaved without a threading requirement.High fidelity DNA replication and repair ensures maintenance of genomic integrity, critical for the viability of eukaryotic cells. Replication on the lagging strand generates short stretches of DNA known as Okazaki fragments that are further processed and finally ligated to form a complete DNA strand. Efficient processing of the Okazaki fragments requires the removal of the RNA/DNA segment that is used to initiate polymerization prior to fragment ligation. Similarly, repair of certain types of DNA damage requires the removal of erroneous or damaged stretches of nucleotides by a process known as long patch-base excision repair (LP-BER).3 Removal of the initiator segment in Okazaki fragment maturation and damaged bases in LP-BER are done by displacing the downstream DNA segment into a 5Ј flap structure by replication or repair-associated polymerases (1).Flap endonuclease 1 (FEN1) is a critical central component of both the replication and repair pathways (1-3). FEN1 is a structure-specific nuclease that recognizes and processes 5Ј flap intermediates displaced by replication and repair associated polymerases (1-4). Biochemical analysis shows that FEN1 possesses endonuclease activity and a minor 5Ј exonuclease function (5, 6). FEN1 is able to recognize and cleave at the base of the flap, effectively creating a nicked DNA segment. Multiple reports have shown that 5Ј flap-bound proteins, annealed DNA segments complementary to the 5Ј flap, or large adducts bound to the 5Ј flap block FEN1 cleavage in vitro (5,(7)(8)(9)(10)(11)(12)(13)(14). Based on results from these 5Ј flap blocking experiments, our group proposed a model that FEN1 must first recognize the 5Ј end of the flap and track down the unblocked single-stranded 5Ј flap before cleaving (7). The steps taken in this tracking model are described in the discussion section. We proposed that the evolutionary development of flap tracking prohibits FEN1 from erroneousl...

SV40 chromosomes undergoing transcription operationally defined by the presence of RNA Polymerase II (RNAPII) were immune-selected with antibody to RNAPII and subjected to secondary chromatin immunoprecipitation with antibodies to hyperacetylated or unacetylated H4 or H3. Immune Selection Fragmentation and Immunoprecipitation (ISFIP) was used to determine the hyperacetylation status of histones independent of the location of the RNAPII and ReChromatin Immunoprecipitation (ReChIP) was used to determine their hyperacetylation status when associated with RNAPII. While hyperacetylated H4 and H3 were found in the coding regions regardless of the location of RNAPII, unacetylated H4 and H3 were only found at sites lacking RNAPII. The absence of unacetylated H4 and H3 at sites containing RNAPII was correlated with the specific association of the Histone Acetyl Transferase (HAT) p300 with the RNAPII. In contrast, the presence of unacetylated H4 and H3 at sites lacking RNAPII was shown to result from the action of a histone deacetylase (HDAC) based upon the effects of the inhibitor sodium butyrate. These results suggest that the extent of hyperacetylation of H4 and H3 during transcription alternates between hyperacetylation directed by an RNAPII associated HAT and deacetylation directed by an HDAC at other sites.

DNA tumor viruses including members of the polyomavirus, adenovirus, papillomavirus, and herpes virus families are presently the subject of intense interest with respect to the role that epigenetics plays in control of the virus life cycle and the transformation of a normal cell to a cancer cell. To date, these studies have primarily focused on the role of histone modification, nucleosome location, and DNA methylation in regulating the biological consequences of infection. Using a wide variety of strategies and techniques ranging from simple ChIP to ChIP-chip and ChIP-seq to identify histone modifications, nuclease digestion to genome wide next generation sequencing to identify nucleosome location, and bisulfite treatment to MeDIP to identify DNA methylation sites, the epigenetic regulation of these viruses is slowly becoming better understood. While the viruses may differ in significant ways from each other and cellular chromatin, the role of epigenetics appears to be relatively similar. Within the viral genome nucleosomes are organized for the expression of appropriate genes with relevant histone modifications particularly histone acetylation. DNA methylation occurs as part of the typical gene silencing during latent infection by herpesviruses. In the simple tumor viruses like the polyomaviruses, adenoviruses, and papillomaviruses, transformation of the cell occurs via integration of the virus genome such that the virus's normal regulation is disrupted. This results in the unregulated expression of critical viral genes capable of redirecting cellular gene expression. The redirected cellular expression is a consequence of either indirect epigenetic regulation where cellular signaling or transcriptional dysregulation occurs or direct epigenetic regulation where epigenetic cofactors such as histone deacetylases are targeted. In the more complex herpersviruses transformation is a consequence of the expression of the viral latency proteins and RNAs which again can have either a direct or indirect effect on epigenetic regulation of cellular expression. Nevertheless, many questions still remain with respect to the specific mechanisms underlying epigenetic regulation of the viruses and transformation.