Biomedical subjects

Tapan Biswas

Publications and source records attributed to Tapan Biswas.

7 recordsLinked to original sources

PubMed2006

Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site.

The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.

Algorithms↗

PubMed2005

DNA arms do the legwork to ensure the directionality of lambda site-specific recombination.

The integrase protein of bacteriophage lambda (Int) catalyzes site-specific recombination between lambda phage and Escherichia coli genomes. Int is a tyrosine recombinase that binds to DNA core sites via a C-terminal catalytic domain and to a collection of arm DNA sites, distant from the site of recombination, via its N-terminal domain. The arm sites, in conjunction with accessory DNA-bending proteins, provide a means of regulating the efficiency and directionality of Int-catalyzed recombination. Recent crystal structures of lambda Int tetramers bound to synaptic and Holliday junction intermediates, together with new biochemical data, suggest a mechanism for the allosteric control of the recombination reaction through arm DNA binding interactions.

Bacteriophage lambda↗

PubMed2005

A structural basis for allosteric control of DNA recombination by lambda integrase.

Site-specific DNA recombination is important for basic cellular functions including viral integration, control of gene expression, production of genetic diversity and segregation of newly replicated chromosomes, and is used by bacteriophage lambda to integrate or excise its genome into and out of the host chromosome. lambda recombination is carried out by the bacteriophage-encoded integrase protein (lambda-int) together with accessory DNA sites and associated bending proteins that allow regulation in response to cell physiology. Here we report the crystal structures of lambda-int in higher-order complexes with substrates and regulatory DNAs representing different intermediates along the reaction pathway. The structures show how the simultaneous binding of two separate domains of lambda-int to DNA facilitates synapsis and can specify the order of DNA strand cleavage and exchange. An intertwined layer of amino-terminal domains bound to accessory (arm) DNAs shapes the recombination complex in a way that suggests how arm binding shifts the reaction equilibrium in favour of recombinant products.

Allosteric Regulation↗

PubMed2005

Architecture of recombination intermediates visualized by in-gel FRET of lambda integrase-Holliday junction-arm DNA complexes.

Lambda integrase (Int) mediates recombination between attachment sites on phage and Escherichia coli DNA. Int is assisted by accessory protein-induced DNA loops in bridging pairs of distinct "arm-type" and "core-type" DNA sites to form synapsed recombination complexes that subsequently recombine by means of a Holliday junction (HJ) intermediate. An in-gel FRET assay was developed and used to measure 15 distances between six points in two Int-HJ complexes containing arm-DNA oligonucleotides, and 3D maps of these complexes were derived by distance-geometry calculations. The maps reveal unexpected positions for the arm-type DNAs relative to core sites on the HJ and a new Int conformation in the HJ tetramer. The results show how the position of arm DNAs determines the bias of catalytic activities responsible for directional resolution, provide insights into the organization of Int higher-order complexes, and lead to models of the structure of the full HJ recombination intermediates.

Bacteriophage lambda↗

PubMed2003

Arm sequences contribute to the architecture and catalytic function of a lambda integrase-Holliday junction complex.

lambda integrase (Int) mediates recombination between attachment sites on lambda phage and E. coli DNAs. With the assistance of accessory proteins that induce DNA loops, Int bridges pairs of distinct arm- and core-type DNA binding sites to form synapsed recombination complexes, which then recombine via a Holliday junction (HJ) intermediate. We show that, in addition to promoting the proper positioning of Int protomers, the arm sequences facilitate the catalytic activities of the Int tetramer, independent of accessory proteins or physical continuity between the arm and core sites. We have determined the architecture of ternary complexes containing a HJ, Int, and P'1,2 arm-type DNA. These structures accommodate simultaneous binding of Int to direct-repeat arm sites and indirect-repeat core sites and afford a new view of the higher-order recombinogenic complexes.

Bacteriophage lambda↗

PubMed2002

Attenuating functions of the C terminus of lambda integrase.

The tyrosine family site-specific recombinases, in contrast to the related type I topoisomerases, which act as monomers on a single DNA molecule, rely on multi-protein complexes to synapse partner DNAs and coordinate two sequential strand exchanges involving four nicking-closing reactions. Here, we analyze three mutants of the catalytic domain of lambda integrase (Int), A241V, I353M and W350ter that are defective for normal recombination, but possess increased topoisomerase activity. The mutant enzymes can carry out individual DNA strand exchanges using truncated substrates or Holliday junctions, and they show more DNA-cleavage activity than wild-type Int on isolated att sites. Structural modeling predicts that the substituted residues may destabilize interactions between the C-terminal beta-strand (beta7) of Int and the core of the protein. The cleavage-competent state of Int requires the repositioning of the nucleophile (Y342) located on beta6 and the catalyst K235 located on the flexible beta2-beta3 loop, relative to their positions in a crystal structure of the inactive conformation. We propose that the anchoring of beta7 against the protein core restrains the movement of Tyr342 and/or Lys235, causing an attenuation of cleavage activity in most contexts. Within a bona fide recombination complex, the release of strand beta7 would allow Tyr342 and Lys235 to assume catalytically active conformations in coordination with other Int protomers in the complex. The loss of beta7 packing by misalignment or truncation in the mutant proteins described here causes a loss of regulated activity, thereby favoring DNA cleavage activity in monomeric complexes and forfeiting the coordination of strand-exchange necessary for efficient recombination.

Amino Acid Sequence↗

PubMed2002

Binding of specific DNA base-pair mismatches by N-methylpurine-DNA glycosylase and its implication in initial damage recognition.

Most DNA glycosylases including N-methylpurine-DNA glycosylase (MPG), which initiate DNA base excision repair, have a wide substrate range of damaged or altered bases in duplex DNA. In contrast, uracil-DNA glycosylase (UDG) is specific for uracil and excises it from both single-stranded and duplex DNAs. Here we show by DNA footprinting analysis that MPG, but not UDG, bound to base-pair mismatches especially to less stable pyrimidine-pyrimidine pairs, without catalyzing detectable base cleavage. Thermal denaturation studies of these normal and damaged (e.g. 1,N(6)-ethenoadenine, varepsilonA) base mispairs indicate that duplex instability rather than exact fit of the flipped out damaged base in the catalytic pocket is a major determinant in the initial recognition of damage by MPG. Finally, based on our determination of binding affinity and catalytic efficiency we conclude that the initial recognition of substrate base lesions by MPG is dependent on the ease of flipping of the base from unstable pairs to a flexible catalytic pocket.

Animals↗