Chapter VI: DNA Replication: Year III Pharm.D Dr.V. Chitra

Chapter VI: DNA Replication: Year III Pharm.D Dr.V. Chitra I. The Molecular Mechanism of DNA Replication „ The copying process of DNA is related t...
Author: Morris Ball
5 downloads 0 Views 381KB Size
Chapter VI: DNA Replication:

Year III Pharm.D Dr.V. Chitra

I. The Molecular Mechanism of DNA Replication „ The copying process of DNA is related

to nitrogenous base pairing rules – Parent DNA molecule consists of two strands ™Complementary – A pairs with T; G pairs with C ™The two strands run in antiparallel directions (DNA has polarity)

– The two DNA strands separate and serve as templates to direct the synthesis of “new” complementary strands – New nucleotides are inserted along the template – A pairs with T; G pairs with C

– Each nucleotide that is added is covalently attached to the previous one ™ Enzyme = DNA Polymerase ™ Sugar-phosphate backbone of new strand is formed „ The linear DNA sequence exists in many states – Each gene has its own UNIQUE sequence – Knowing the sequence of one strand, you can deduce the sequence of the other (complementarity)

II. Three Models of DNA Replication „ Conservative model

– Parent molecule remains the same – Completely new copy of the double helix is made „ Semiconservative model – Parent strands separate and serve as templates for new strand synthesis – Hybrid molecules are made „ Dispersive model – New strands contain a mixture of old molecules and newly synthesized molecules

„ Messelson and Stahl Experiment

Supports the Semi-Conservative Model of DNA Replication

III. DNA Replication Involves a Complex Assembly of Proteins and Enzymes „ Human haploid genome = 3 x 109 bp

– ~>1000 X more complex than Escherichia coli – The Human Genome Project has sequenced the entire genome of our species ™Worldwide effort – International Collaborations

– The DNA synthesis phase (interphase) during mitosis lasts only a few hours despite its huge size – Replication of the DNA sequence is very accurate ™Mutation rate ~1/109 errors

The Sequence of Events 1. Beginning of replication occurs at the origins of replication – Prokaryotic cells (e.g. E. coli) contain only one origin – Eukaryotic cells contain thousands of orgins on each chromosome – Proteins bind to origins and pry open the two strands

– A replication bubble appears at the site of strand separation and new DNA synthesis ™Replication forks forma at the ends of the bubbles – Replication occurs in both directions on the two strands ™But ALWAYS in the 5’ Æ 3’ direction per strand – Replication bubbles fuse

DNA Replication „ Origins of replication

1. Replication Forks: hundreds of Y-shaped regions of replicating DNA molecules where new strands are growing. 3’

5’ Parental DNA Molecule 3’

Replication Fork 5’

DNA Replication „ Origins of replication

2. Replication Bubbles: a. Hundreds of replicating bubbles (Eukaryotes). b. Single replication fork (bacteria). Bubbles



DNA polymerases add on new nucleotides to the growing DNA strand – One nucleotide is added at a time to the 3’-OH group of the previous nucleotide – The 3’-OH group of the ribose sugar is covalently linked to the nucleoside triphosphate forming a phosphodiester bond – Two phosphate groups are liberated – energy is released (PPi – pyrophosphate)

3. How to resolve the replication fork

dilemma of antiparallel strands – DNA strands have polarity (antiparallel) – DNA polymerase can only add new nucleotides to the 3’ end of the terminal deoxyribose – Synthesis always progresses in the 5’Æ 3’ direction

– Leading strand is synthesized continuously ™DNA polymerase progresses as DNA is unzipped ™One continuous polymer is made

– Lagging strand is synthesized discontinuously ™Synthesized in opposite direction ™Synthesized AWAY from the replication fork ™Initiated as a series of short segments called Okazaki fragments (100-200 bases long) ™Okazaki fragments are joined together by DNA ligase (covalent phosphodiester bonds between fragments)


RNA primers are required for initiation of DNA synthesis – DNA polymerase can add nucleotides only to 3’OH group of an already existing nucleotide paired to its complement on the other strand – Q: How do things get started? – A: RNA primers are made by an enzyme called PRIMASE ™ ~10 nucleotides long primers Æ H-bonds to template and provides substitute for DNA polymerase


strand requires only one RNA primer ™Lagging strand requires one RNA primer for every Okazaki fragment – RNA primers are removed by specific enzymes and replaced with DNA nucleotides ™Gaps are sealed with DNA ligase


Helicases and single-stranded binding proteins are important components of DNA synthesis – Helicases unwind the double helix and separate the two templates ™ Smoothing of twists ™ Breaking of H-bonds – Single-stranded binding proteins stabilize the DNA for replication

The Telomere Problem „ Eukaryotic cells have a problem replicating the 5’ ends of

daughter DNA strands – The ends of chromosomes contain 100-1000 repeating (TTAGGG)n segments – Result: Ends of DNA get shorter and shorter in most dividing somatic cells ™ Older people have shorter telomeres „ Some cells have a solution: – Telomerase ™ Enzyme + RNA fragment – catalyzes the extension of the ends ™ RNA = template for new telomere pieces – Examples: ™ Germ cells ™ Some cancerous cells ™ Immortalized cultured cells