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

G0
Quiescent cells

G1
phase

phase

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

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, synthesis of one of the strands... 5 3

...has to be discontinuous.
This is the lagging strand.

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

RNA primer Okazaki fragment

RNA primer

RNA primer 5

pol III 5 DNA polymerase III initiates at the primer and elongates DNA up to the next RNA primer

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

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

Properties of DNA polymerases

DNA polymerases of E. 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, 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

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