Structure of DNA DNA contains deoxyribonucleotides linked covalently by 3'  5‘ phosphodiester linkage Deoxyribonucleases cleave phosphodiester linkage in DNA and ribonucleases cleave phosphodiester linkage in RNA DNA strands wind around each other forming double helix ( a twisted ladder) with a common axis of symmetry DNA strands are paired in an antiparallel manner, that is 5‘ end of one strand is paired to 3‘ end of the other strand The hydrophilic deoxyribose-phosphate backbones of both strands are on the outside and the hydrophobic stacking bases are on the inside

Structure of DNA The spatial relationship between the two strands in the helix creates a major (wide) groove and a minor (narrow) groove The base sequence in one strand is complementary to the base sequence in the other strand The total amount of purines is equal to the total amount of pyrimidines These base pairs are perpendicular to the axis of symmetry The base pairs are held by hydrogen bonding; two between A and T and three between G and C These hydrogen bonds, plus the hydrophobic interactions between the stacked bases, stabilise the structure of the double helix

Separation of to DNA strands in the double helix When hydrogen bonds between paired bases are disrupted, the two strands of the double helix separate Melting temperature is the temperature at which one half of the DNA double helix is lost (half dsDNA  ssDNA) DNA denaturation is monitored at 260nm, where ssDNA has higher absorbance than dsDNA DNA that contains high AT concentration melts (denatures) at lower temperature than GC rich DNA Complementary DNA strands can reform the double helix in annealling or renaturation

DNA replication 1.Two strands of DNA double helix separate 2.Each serve as template for replication of new complementary antiparallel strand 3.Two daughter DNA duplexes; each contains individual parental strand  semi-conservative replication, where parental duplex is not conserved as entity, instead it’s present in daughter duplexes Initiation of replication requires prepriming complex to 1.Recognize origin of replication-single in prokaryores and multiple in euokaryotes ( by dnaA protein) 2.Maintain separation of parental ssDNA ( by SSB proteins) 3.Unwind dsDNA ahead of replication fork –bidirectional ( by DNA helicase)

DNA replication Topoisomerases I and II remove the supercoils accumulating ahead of replication forks Elongation of daughter DNA growing strands Two newly synthesized strands must grow in opposite directions; one in 5  3 direction towards the replication fork ( Leading strand, synthesized continuously) and one in 5  3 direction away from replication fork (lagging strand, synthesized discontinuously as in Okazaki fragments) DNA elongation requires 1.RNA polymerase-Primase: synthesizes 10ribonucleotide long RNA which is complementary antiparallel to ssDNA template. One RNA primer at the origin of replication is required for the leading strand, and multiple RNA primers are synthesized at replication fork for the lagging strand

DNA replication DNA elongation requires 2.DNA polymerase III (3  5 reading, 5  3polymerase and 3  5 exonuclease for proofreading)Daughter DNA strands elongation is catalysed by DNA polymerase III, using the 3OH end of RNA primer as first acceptor of dNMP addition, specified by the base sequence of the parental ssDNA template. 3.DNA polymearse I (5  3 exonuclease, 5  3polymerase and 3  5 exonuclease for proofreading) it locates on the lagging strand the space between 3end of Okazaki fragment-synthesized by polymerase III and 5end phosphate group of adjacent RNA primer, and carries removal/synthesis/proofreading one nucleotide a time until RNA primer degraded and replaced by DNA

DNA Replication DNA elongation requires 4.DNA ligase; catalyses the final phosphodiester linkage between 5end of DNA Okazaki fragment synthesized by polymerase III and 3 end of DNA chain synthesized by polymerase I – replacing the RNA primer Termination of replication 1. Ter (terminus ) region of E.coli chromosome, which is rich with Gs and Ts, signals end of replication 2.Tus ( Terminator Utilisation Substance) is a protein that binds the Ter region, inhibiting helicase activity and, thus, preventing the replication fork