DNA Replication

i). Cell cycle/ semi-conservative replication ii). Initiation of DNA replication iii). Discontinuous DNA synthesis iv). Components of the replication apparatus BTY 885 Sept. 03rd, 2011

The Plant cell cycle Rapid growth and preparation for DNA synthesis DNA synthesis and histone synthesis phase S G0 Quiescent cells G1 phase phase M phase G2 Growth and preparation for cell division Mitosis .

DNA replication is semi-conservative Parental DNA strands Each of the parental strands serves as a template for a daughter strand Daughter DNA strands .

Origins of DNA replication on mammalian chromosomes origins of DNA replication (every ~150 kb) 5’ 3’ 3’ 5’ bidirectional replication replication bubble fusion of bubbles 5’ 3’ 5’ 3’ daughter chromosomes 3’ 5’ 3’ 5’ .

DNA polymerase 5’ 5’ RNA primer 3’ 5’ newly synthesized DNA 3’ .

. 5’ 3’ .Discontinuous synthesis of DNA 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ Because DNA is always synthesized in a 5’ to 3’ direction. . This is the lagging strand..has to be discontinuous.. synthesis of one of the strands..

Each replication fork has a leading and a lagging strand leading strand (synthesized continuously) replication fork 5’ 3’ 3’ 5’ replication fork 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ lagging strand (synthesized discontinuously) • The leading and lagging strand arrows show the direction of DNA chain elongation in a 5’ to 3’ direction • The small DNA pieces on the lagging strand are called Okazaki fragments (100-1000 bases in length) .

RNA primer direction of leading strand synthesis 3’ 5’ replication fork 5’ 3’ 3’ 5’ direction of lagging strand synthesis .

Strand separation at the replication fork causes positive supercoiling of the downstream double helix 3’ 5’ 5’ 3’ 3’ 5’ • DNA gyrase is a topoisomerase II. which breaks and reseals the DNA to introduce negative supercoils ahead of the fork • Fluoroquinolone antibiotics target DNA gyrases in many gram-negative bacteria: ciprofloxacin and levofloxacin (Levaquin) .

Movement of the replication fork 5’ 3’ 5’ 3’ .

Movement of the replication fork 5’ RNA primer Okazaki fragment RNA primer .

3’ RNA primer 5’ pol III 5’ DNA polymerase III initiates at the primer and elongates DNA up to the next RNA primer 3’ 5’ 5’ 3’ newly synthesized DNA (100-1000 bases) (Okazaki fragment) 5’ pol I DNA polymerase I inititates at the end of the Okazaki fragment and further elongates the DNA chain while simultaneously removing the RNA primer with its 5’ to 3’ exonuclease activity .

3’ 5’ newly synthesized DNA (Okazaki fragment) DNA ligase seals the gap by catalyzing the formation of a 3’. 5’-phosphodiester bond in an ATP-dependent reaction 3’ 5’ .

coli_ Polymerization: 5’ to 3’ Proofreading exonuclease: 3’ to 5’ Repair exonuclease: 5’ to 3’ pol I pol II pol III (core) yes yes yes yes yes yes yes no no DNA polymerase III is the main replicating enzyme DNA polymerase I has a role in replication to fill gaps and excise primers on the lagging strand.Properties of DNA polymerases DNA polymerases of E. and it is also a repair enzyme and is used in making recombinant DNA molecules • all DNA polymerases require a primer with a free 3’ OH group • all DNA polymerases catalyze chain growth in a 5’ to 3’ direction • some DNA polymerases have a 3’ to 5’ proofreading activity .

Cell cycle/ semi-conservative replication ii). Initiation of DNA replication iii). Discontinuous DNA synthesis iv). 03rd. 2011 . Components of the replication apparatus BTY 885 Sept.DNA Replication i).