16.2 DNA Replication

Layout of the Eukaryote DNA Two DNA strands are antiparallel Run in opposite directions 3’ (three prime) – 5’ (five prime) 5’ (five prime) – 3’ (three prime)

DNA Replication Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material

DNA Replication Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

Fig. 16-9-1 A T C G T A A T G C (a) Parent molecule Figure 16.9 A model for DNA replication: the basic concept

(b) Separation of strands Fig. 16-9-2 A T A T C G C G T A T A A T A T G C G C (a) Parent molecule (b) Separation of strands Figure 16.9 A model for DNA replication: the basic concept

(b) Separation of strands Fig. 16-9-3 A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of strands (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand Figure 16.9 A model for DNA replication: the basic concept

Semiconservative Model Each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand

(a) Conservative model Fig. 16-10 First replication Second replication Parent cell (a) Conservative model (b) Semiconserva- tive model Figure 16.10 Three alternative models of DNA replication (c) Dispersive model

Matthew Meselson and Franklin Stahl

DNA in Prokaryotes and Eukaryotes ring of chromosome holds nearly all of the cell’s genetic material

Prokaryote DNA Replication DNA replication begins at a single point and continues to replicate whole circular strand Replication goes in both directions around the DNA (begins with replication fork)

Fig. 16-12 Origin of replication Parental (template) strand Daughter (new) strand Replication fork Double- stranded DNA molecule Replication bubble 0.5 µm Two daughter DNA molecules (a) Origins of replication in E. coli Origin of replication Double-stranded DNA molecule Parental (template) strand Figure 16.12 Origins of replication in E. coli and eukaryotes Daughter (new) strand 0.25 µm Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes

DNA Replication Overview DNA splits into two strands Complementary base pairs fill in (A with T, C with G) Left with two DNA molecules Semiconservative model Identical

Eukaryote DNA Replication Begins in hundreds of locations along the chromosome Origins of replication

Initiation of DNA Replication Begins when the DNA molecule “unzips” Replication fork Replication “bubble” Hydrogen bonds between base pairs breaks Helicase Single-strand binding proteins Topoisomerase – relieves pressure of DNA ahead of replication fork

Single-strand binding proteins Fig. 16-13 Primase Single-strand binding proteins 3 Topoisomerase 5 3 RNA primer Figure 16.13 Some of the proteins involved in the initiation of DNA replication 5 5 3 Helicase

Synthesis of a New DNA Strand Each strand serves as a template for a new strand to form Primer of RNA The primer is short (5–10 nucleotides long), and the 3 end serves as the starting point for the new DNA strand Complimentary bases will attach DNA polymerase E. coli – DNA polymerase III and DNA polymerase I Humans – 11 different DNA polymerase molecules

Synthesis of a New DNA Strand RNA primer Nucleoside triphosphate As each nucleotide is added to the new strand, 2 phosphates are lost Hydrolysis releases energy to drive reaction

Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate dATP supplies adenine to DNA and is similar to the ATP of energy metabolism The difference is in their sugars: dATP has deoxyribose while ATP has ribose As each monomer of dATP joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Synthesis of a New DNA Strand Antiparallel Elongation Remember 3’ – 5’ and 5’ – 3’ Replication in the 3’ to 5’ direction ONLY MEANING the NEW strand of DNA will form starting with the 5’ end Leading strand (only 1 primer needed – moves toward the replication fork) Lagging strand (many primers needed – moves away from replication fork)

Fig. 16-16b1 Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand Figure 16.6 Synthesis of the lagging strand

Fig. 16-16b2 Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 Figure 16.6 Synthesis of the lagging strand

Fig. 16-16b3 Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 3 Okazaki fragment 5 3 1 5 Figure 16.6 Synthesis of the lagging strand

Fig. 16-16b4 Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 3 Okazaki fragment 5 3 5 1 3 5 3 2 1 5 Figure 16.6 Synthesis of the lagging strand

Fig. 16-16b5 Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 3 Okazaki fragment 5 3 5 1 3 5 3 2 1 5 5 3 Figure 16.6 Synthesis of the lagging strand 3 5 2 1

Fig. 16-16b6 Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 3 Okazaki fragment 5 3 5 1 3 5 3 2 1 5 5 3 Figure 16.6 Synthesis of the lagging strand 3 5 2 1 5 3 3 1 5 2 Overall direction of replication

Important Enzymes Helicase, single-strand binding protein, topoisomerase Primase Synthesis of RNA primer DNA polymerase III (DNA pol III) Add new bases to DNA strand DNA polymerase I (DNA pol I) Removes and replaces RNA primer from 5’ end DNA ligase Links Okazaki fragments and replaces RNA primer from 3’ end

Topoisomerase Video http://www.youtube.com/watch?v=EYGrElVyHnU

The Finished Product Each DNA molecule has one original strand and one new strand Molecules are identical

DNA Replication Video http://www.youtube.com/watch?v=teV62zrm2P0

Fig. 16-UN5

Repair of DNA DNA polymerase Nuclease Proofreads and repairs damaged/mismatched DNA Nuclease Removes section of DNA that is damaged DNA polymerase and DNA ligase replace missing portion

Telomeres Found at the ends of each chromosome Contain no genes Sequence that can be cut short and will not affect normal functioning TTAGGG Telomerase lengthens telomeres in gametes Aging? Cancer? As telomeres get shorter, aging may be triggered. Telomeres shorten and may start affecting replication (cancer cells) – trigger cell death - apoptosis

Telomeres The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist

16.3 A chromosome consists of a DNA molecule packed together with proteins

Chromosomes

Chromosome Structure Bacterial chromosome Eukaryotic chromosomes double-stranded circular small amount of protein Eukaryotic chromosomes Linear DNA molecules large amount of protein DNA in bacteria is “supercoiled” and found in a region of the cell called the nucleoid

Chromatin and Histones Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells Histones are proteins that are responsible for the first level of DNA packing in chromatin Form a tight bond because DNA is negatively charged and the histones have a positive charge

Nucleosomes, or “beads on a string” (10-nm fiber) Fig. 16-21a Nucleosome (10 nm in diameter) DNA double helix (2 nm in diameter) H1 Histone tail Histones Figure 16.21a Chromatin packing in a eukaryotic chromosome DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

Looped domains (300-nm fiber) Metaphase chromosome Fig. 16-21b Chromatid (700 nm) 30-nm fiber Loops Scaffold 300-nm fiber Figure 16.21b Chromatin packing in a eukaryotic chromosome Replicated chromosome (1,400 nm) 30-nm fiber Looped domains (300-nm fiber) Metaphase chromosome

Chromosome Organization Chromatin is organized into fibers 10-nm fiber DNA winds around histones to form nucleosome “beads” Nucleosomes are strung together 30-nm fiber Interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber

Chromosome Organization 300-nm fiber The 30-nm fiber forms looped domains that attach to proteins Metaphase chromosome The looped domains coil further The width of a chromatid is 700 nm

Euchromatin Most chromatin is loosely packed in the nucleus during interphase Condenses prior to mitosis Euchromatin

Heterochromatin During interphase, a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions