DNA Replication PDF

DNA REPLICATION Learning outcomes At the end of this topic, students should be able to Explain the process of DNA replication Explain terms such as the origin of DNA replication, replication fork, leading, and Lagging strands, priming of DNA synthesis, proofreading, and torque. Explain th...

DNA REPLICATION Learning outcomes At the end of this topic, students should be able to Explain the process of DNA replication Explain terms such as the origin of DNA replication, replication fork, leading, and Lagging strands, priming of DNA synthesis, proofreading, and torque. Explain the role of initiator protein, helicase enzyme, primase enzyme, SSB proteins, Topoisomerase enzyme, and DNA polymerase enzyme 111 in prokaryotes & DNA polymerase  (alpha)   (delta) in eukaryotes, ligase enzyme.  DNA replication is the synthesis of new DNA molecule from pre- existing DNA. This occurs during cell division (Mitosis & Meiosis) in the Synthesis phase (S phase) of interphase stage. Semiconservative Model of DNA replication This method of DNA replication was proposed by Watson & Crick. According to this model, the two polynucleotide strands unwind and separate, and act as template for the synthesis of new complimentary strand. So each new daughter DNA has one new DNA strand & one parental strand. SEMICONSERVATIVE MODEL  Bacterium E.coli, has a single chromosome of 5 million base pairs. Under favorable conditions E.coli can copy all of this DNA & divide into two daughter cells in less than an hour. Man has 46 DNA molecules in its nucleus, 1 giant DNA molecule per chromosome. It represents more than 3 billion base pairs. Human chromosomes range in size from about 50,000,000 to 300,000,000 base pairs. There are estimated 30,000 genes in the human genome. A cell takes just few hrs to copy all this DNA with very few errors. GETTING STARTED: i) ORIGINS OF REPLICATION Replication begins at specific sites along the DNA molecule called origin of replication. The circular bacterial chromosome has a single origin, a stretch of DNA having a specific sequence of nucleotides. Proteins that initiate DNA replication recognize this sequence and attach to the DNA, separating the two strands & opening up a replication bubble. Replication then proceeds in both directions, until the entire molecule is copied. Bidirectional replication  In contrast eukaryotic chromosomes may have thousands of replication origins. Multiple replication bubbles forms & eventually fuse, thus speeding the copying. DNA replication proceeds in both direction of origin. At each end of replication bubble is a replication fork, a Y-shaped region where the new strands are elongating. REPLICATION FORK ii) INITIATION OF DNA REPLICATION An initiator protein binds to the origin of replication sequence. DNA helicase enzyme then binds to the origin of replication and denatures the hydrogen bond and untwists the double stranded DNA. The two separated DNA strands are called template strands. or Template strand iii) PRIMING OF DNA SYNTHESIS In replication, the start of a new chain is not DNA but a short stretch of RNA, the primer. It is 10 nucleotides long in eukaryotes & is synthesized using primase enzyme. In prokaryotes it is 10-15 base pairs long. This RNA primer is later replaced by DNA nucleotides.  iv) STABILIZATION OF SINGLE STRANDED DNA IN REPLICATION FORK The separated strands have a tendency to form double helix or coil upon itself through intra-molecular base pairing. Molecules of single- strand DNA binding protein (SSB protein) line up along the unpaired DNA strands, holding them apart while they serve as template for the synthesis of new complimentary strands. v) RELIEVING THE TORQUE GENERATED BY UNWINDING For replication to proceed at 1000 nucleotides per second, the parental helix ahead of the replication fork must rotate 100 revolutions per second. To relieve the resulting twisting, called torque, enzymes known as topoisomerases or gyrases cleave a strand of the helix, allow it to swivel around the intact strand, and then reseal the broken strand. vi) ELONGATING A NEW DNA STRAND Elongation of the new strand at a replication fork is catalyzed by DNA polymerase enzyme111 in prokaryotes & DNA polymerase  (alpha)   (delta) in eukaryotes. As nucleotides align along the template strand they are added to the growing end of the new DNA strand at the rate of 500 nucleotides per sec in bacteria & 50 nucleotides per sec in humans. vii) LEADING & LAGGING STRAND ANTIPARALLEL ARRANGEMENT OF DNA STRAND The two DNA strands are antiparallel. DNA polymerase add nucleotide only to the 3’end of a growing nucleotide strand, never to the 5’end. Thus a new DNA strand can elongate only in the 5’-3’ direction. Synthesis of the leading strand  Along the 3’-5’ strand DNA polymerase can synthesize a continuous complimentary strand in the 5’-3’ direction. The DNA strand made by this mechanism is called the leading strand. To elongate the other strand the DNA polymerase enzyme work in the direction away from the replication fork. The DNA synthesized in this manner is called a lagging strand. This strand is synthesized as a series of segments called Okazaki fragments, which is 100- 200 nucleotides long in eukaryotes. DNA ligase enzymes join the sugar–phosphate backbones of the fragments to create a single DNA strand. Note the helicase enzyme (key) breaking the hydrogen bonds, DNA polymerase enzyme (brush) bring in nucleotides to make the new strand and ligase enzyme sealing the okasaki fragments of lagging strand. viii) PROOF READING The DNA polymerase enzyme is constantly checking for incorrect nucleotides. In bacteria, when an incorrect base pair is recognized, DNA polymerases I and II, excises the mismatched base and it is replaced by correct one by the 3’-5’ exonuclease activity. In eukaryotes, DNA polymerase    does the proof reading. DNA REPLICATION DNA REPLICATION DNA REPLICATION