Ross B. Inman

Partial denaturation of thymine- and 5-bromouracil-containing λ DNA in alkali☆

An electron microscopic examination of λ DNA, partially denatured at high pH, shows that the sections which denature first are at the same positions as those previously observed for λ DNA partially denatured by heat. A similar study of BU†-labeled λ DNA shows the A·BU-rich regions to be situated at similar positions along the molecule to the A·T-rich regions in normal λ DNA, but it also shows that the A·BU-rich regions melt out at a lower pH. Details of a modification to the protein film technique are given in the Materials and Methods section.

Position of branch points in replicating λ DNA

Abstract Recent experiments by Ogawa, Tomizawa & Fuke (1968) have shown that it is possible to isolate intracellular λ DNA having a circular structure but with an extra length of DNA extending between two branch points. An attempt has been made to map the position of these branch points with respect to the point on the circular structure corresponding to the ends of the mature λ phage DNA molecule. If the circular branched molecules do, in fact, represent replicating DNA, then certain conclusions may be drawn concerning the starting point and direction of replication.

Recombinational DNA Repair: The RecF and RecR Proteins Limit the Extension of RecA Filaments beyond Single-Strand DNA Gaps

In the presence of both the RecF and RecR proteins, RecA filament extension from a single strand gap into adjoining duplex DNA is attenuated. RecR protein alone has no effect, and RecF protein alone has a reduced activity. The RecFR complexes bind randomly, primarily to the duplex regions of the DNA, and the extension of the RecA filament is halted at the first complex encountered. A very slow lengthening of RecA filaments observed in the presence of RecFR is virtually eliminated when RecF is replaced with an RecF mutant protein that does not hydrolyze ATP. These observations are incorporated into an expanded model for the functions of RecF, RecO, and RecR proteins in the early stages of postreplication DNA repair.

Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes : implications for phage evolution

A118 is a temperate phage isolated from Listeria monocytogenes. In this study, we report the entire nucleotide sequence and structural analysis of its 40 834 bp DNA. Electron microscopic and enzymatic analyses revealed that the A118 genome is a linear, circularly permuted, terminally redundant collection of double‐stranded DNA molecules. No evidence for cohesive ends or for a terminase recognition (pac) site could be obtained, suggesting that A118 viral DNA is packaged via a headful mechanism. Partial denaturation mapping of DNA cross‐linked to the tail shaft indicated that DNA packaging proceeds from left to right with respect to the arbitrary genomic map and the direction of genes necessary for lytic development. Seventy‐two open reading frames (ORFs) were identified on the A118 genome, which are apparently organized in a life cycle‐specific manner into at least three major transcriptional units. N‐terminal amino acid sequencing, bioinformatic analyses and functional characterizations enabled the assignment of possible functions to 26 ORFs, which included DNA packaging proteins, morphopoetic proteins, lysis components, lysogeny control‐associated functions and proteins necessary for DNA recombination, modification and replication. Comparative analysis of the A118 genome structure with other bacteriophages revealed local, but sometimes extensive, similarities to a number of phages spanning a broader phylogenetic range of various low G+C host bacteria, which implies relatively recent exchange of genes or genetic modules. We have also identified the A118 attachment site attP and the corresponding attB in Listeria monocytogenes, and show that site‐specific integration of the A118 prophage by the A118 integrase occurs into a host gene homologous to comK of Bacillus subtilis, an autoregulatory gene specifying the major competence transcription factor.

RecA protein promotes the regression of stalled replication forks in vitro

Replication forks are halted by many types of DNA damage. At the site of a leading-strand DNA lesion, forks may stall and leave the lesion in a single-strand gap. Fork regression is the first step in several proposed pathways that permit repair without generating a double-strand break. Using model DNA substrates designed to mimic one of the known structures of a fork stalled at a leading-strand lesion, we show here that RecA protein of Escherichia coli will promote a fork regression reaction in vitro. The regression process exhibits an absolute requirement for ATP hydrolysis and is enhanced when dATP replaces ATP. The reaction is not affected by the inclusion of the RecO and R proteins. We present this reaction as one of several potential RecA protein roles in the repair of stalled and/or collapsed replication forks in bacteria.

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecA/DnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecA/DnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

DNA Strand Exchange Promoted by RecA K72R TWO REACTION PHASES WITH DIFFERENT Mg REQUIREMENTS

Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C.(1993) J. Biol. Chem. 268, 1292-1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg in ways that reflect the concentration of a Mg•dATP complex. When Mg is present at concentrations just sufficient to form the Mg•dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly). Higher levels of Mg are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg levels that inhibit the subsequent migration of a DNA branch. We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands. The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5′-O-(3-thio)triphosphate. The Mg effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some experiments, are interpreted in the context of a model in which a fast phase of DNA strand exchange produces a discontinuous three-stranded DNA pairing intermediate, followed by a slow phase in which the discontinuities are resolved. The mutant protein also facilitates the autocatalytic cleavage of the LexA repressor, but at a reduced rate.

The RecOR proteins modulate RecA protein function at 5′ ends of single-stranded DNA

The Escherichia coli RecF, RecO and RecR pro teins have previously been implicated in bacterial recombinational DNA repair at DNA gaps. The RecOR‐facilitated binding of RecA protein to single‐stranded DNA (ssDNA) that is bound by single‐stranded DNA‐binding protein (SSB) is much faster if the ssDNA is linear, suggesting that a DNA end (rather than a gap) facilitates binding. In addition, the RecOR complex facilitates RecA protein‐mediated D‐loop formation at the 5′ ends of linear ssDNAs. RecR protein remains associated with the RecA filament and its continued presence is required to prevent filament disassembly. RecF protein competes with RecO protein for RecR protein association and its addition destabilizes RecAOR filaments. An enhanced function of the RecO and RecR proteins can thus be seen in vitro at the 5′ ends of linear ssDNA that is not as evident in DNA gaps. This function is countered by the RecF/RecO competition for association with the RecR protein.

Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed + 1 translational frameshifting in structural protein synthesis

PSA is a temperate phage isolated from Listeria monocytogenes strain Scott A. We report its complete nucleotide sequence, which consists of a linear 37 618 bp DNA featuring invariable, 3′‐protruding single stranded (cohesive) ends of 10 nucleotides. The physical characteristics were confirmed by partial denaturation mapping and electron microscopy of DNA molecules. Fifty‐seven open reading frames were identified on the PSA genome, which are apparently organized into three major transcriptional units, in a life cycle‐specific order. Functional assignments could be made to 33 gene products, including structural proteins, lysis components, DNA packaging proteins, lysogeny control functions and replication proteins. Bioinformatics demonstrated relatedness of PSA to phages infecting lactic acid bacteria and other low G + C Gram‐positives, but revealed only few similarities to Listeria phage A118. Virion proteins were analysed by amino acid sequencing and mass spectrometry, which enabled identification of major capsid and tail proteins, a tape measure and a putative portal. These analyses also revealed an unusual form of translational frameshifting, which occurs during decoding of the mRNAs specifying the two major structural proteins. Frameshifting yields different length forms of Cps (gp5) and Tsh (gp10), featuring identical N‐termini but different C‐termini. Matrix‐assisted laser‐desorption ionization mass spectrometry (MALDI‐MS) and electrospray ionization mass spectrometry (ESI‐MS) of tryptic peptide fragments was used to identify the modified C‐termini of the longer protein species, by demonstration of specific sequences resulting from + 1 programmed translational frameshifting. A slippery sequence with overlapping proline codons near the 3′ ends of both genes apparently redirects the ribosomes and initiates the recoding event. Two different cis‐acting factors, a shifty stop and a pseudoknot, presumably stimulate frameshifting efficiency. PSA represents the first case of + 1 frameshifting among dsDNA phages, and appears to be the first example of a virus utilizing a 3′ pseudoknot to stimulate such an event.

Denaturation maps of the left and right sides of the lambda DNA molecule determined by electron microscopy

Partially denatured λ DNA has been examined in the electron microscope and the position of a large number of denatured sites located and expressed in the form of denaturation maps. Between 12 and 15 regions have been found where denatured sites occur with high frequency.