Free energy minimization has been the most popular method for RNA secondary structure prediction for decades. It is based on a set of empirical free energy change parameters derived from experiments using a nearest-neighbor model. In this study, a program, MaxExpect, that predicts RNA secondary structure by maximizing the expected base-pair accuracy, is reported. This approach was first pioneered in the program CONTRAfold, using pair probabilities predicted with a statistical learning method. Here, a partition function calculation that utilizes the free energy change nearest-neighbor parameters is used to predict basepair probabilities as well as probabilities of nucleotides being single-stranded. MaxExpect predicts both the optimal structure (having highest expected pair accuracy) and suboptimal structures to serve as alternative hypotheses for the structure. Tested on a large database of different types of RNA, the maximum expected accuracy structures are, on average, of higher accuracy than minimum free energy structures. Accuracy is measured by sensitivity, the percentage of known base pairs correctly predicted, and positive predictive value (PPV), the percentage of predicted pairs that are in the known structure. By favoring doublestrandedness or single-strandedness, a higher sensitivity or PPV of prediction can be favored, respectively. Using MaxExpect, the average PPV of optimal structure is improved from 66% to 68% at the same sensitivity level (73%) compared with free energy minimization.

Mitogen-activated protein kinase kinase (MKK) phosphorylates and activates mitogen-activated protein kinase (MAPK) in response to stimulation of various eukaryotic signaling pathways. Conversely, a recent report showed that MAPK phosphorylates MKK in vitro [Matsuda, S., Gotoh, Y., and Nishida, E. (1993) J. Biol. Chem. 268, 3277-3281]. To gain insight into the function of this feedback phosphorylation, we identified the major sites targeted for phosphorylation by MAPK and examined whether such a modification plays a role in regulating the basal and stimulated MKK activities. Two phosphopeptides generated by tryptic digestion of MAPK-phosphorylated MKK were identified by electrospray ionization mass spectrometry. Cyanogen bromide cleavage also yielded two phosphopeptides whose sequence overlapped with the tryptic phosphopeptides. Both sets of phosphopeptides contained candidate MAPK target sites at Thr292 and Thr386 that fit the consensus sequence ProXThr*Pro. Replacement of either Thr292 or Thr386 with alanine by site-directed mutagenesis reduced the phosphate incorporation respectively to 32 or 75% that of wild type MKK. Replacement of both threonine residues with alanine reduced phosphate incorporation to 2.5% that of wild type enzyme. Comparison of MAPK-phosphorylated vs. unphosphorylated MKK showed no significant differences in basal or Raf-1-stimulated MKK activity. We conclude that the phosphorylation of MKK at Thr292 and Thr386 does not interfere with catalysis in vitro.

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...

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

Eukaryotic DNA replication is a complex process requiring the proper functioning of a multitude of proteins to create error-free daughter DNA strands and maintain genome integrity. Even though synthesis and joining of Okazaki fragments on the lagging strand involves only half the DNA in the nucleus, the complexity associated with processing these fragments is about twice that needed for leading strand synthesis. Flap endonuclease 1 (FEN1) is the central component of the Okazaki fragment maturation pathway. FEN1 cleaves flaps that are displaced by DNA polymerase δ (pol δ), to create a nick that is effectively joined by DNA ligase I. The Pif1 helicase and Dna2 helicase/ nuclease contribute to the maturation process by elongating the flap displaced by pol δ. Though the reason for generating long flaps is still a matter of debate, genetic studies have shown that Dna2 and Pif1 are both important components of DNA replication. Our current knowledge of the exact enzymatic steps that govern Okazaki fragment maturation has heavily derived from reconstitution reactions in vitro, which have augmented genetic information, to yield current mechanistic models. In this review we describe both the design of specific DNA substrates that simulate intermediates of fragment maturation and protocols for reconstituting partial and complete lagging strand replication.