Lecture 24: DNA replication G 0.34 nm 3.4 nm 1 nm Figure 16.7a, c (c) Space-filling model

Lecture Outline Overview of DNA replication DNA has polarity (5’P, 3’OH) Rules of Replication Details of the replication machine Proofreading Mutation Special problems at the ends of chromosomes C T A G 0.34 nm 3.4 nm 1 nm

Review Watson-Crick model of DNA structure Base composition ratios G 0.34 nm 3.4 nm 1 nm Watson-Crick model of DNA structure Base composition ratios A=T; G=C Radioactive labeling experiments show that DNA is the genetic material Replication is semi-conservative

Chromosomes Bacterial chromosomes tend to be circular, eukaryotic chromosomes linear DNA plus associated proteins = chromatin Euchromatin vs heterochromatin

DNA Structure N H O CH3 Sugar Adenine (A) Thymine (T) Guanine (G) 0.34 nm 3.4 nm 1 nm N H O CH3 Sugar Adenine (A) Thymine (T) Guanine (G) Cytosine (C) Figure 16.8 5’ Carbon has PO4 3’ Carbon has OH

In DNA replication The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. A C T G Figure 16.9 a–d

Replication overview Must maintain integrity of the DNA sequence through successive rounds of replication (think of the game “telephone”) Need to: unwind DNA, add an RNA primer, find an appropriate base, add it to the growing DNA fragment, proofread, remove the initial primer, fill in the gap with DNA, ligate fragments together All of this is fast, about 100 bp/second animation from DNAi

Elongating a New DNA Strand DNA polymerases, add nucleotides to the 3 end of a growing strand New strand Template strand 5 end 3 end Sugar A T Phosphate Base C G G C A T P P P C OH Figure 16.13 5 end Nucleoside triphosphate P P Pyrophosphate

Template 3’ 5’ 3’ 5’ Primer with a free 3’ OH P C OH T G A P G P T P A

Rules of Replication Semi-conservative Primer is required Template is required Elongation occurs 5’ -> 3’  Semi-discontinuous Bidirectional

DNA synthesis Needs a template Must add a base to an existing 3’ OH This end has a 5’ phosphate Needs a template Must add a base to an existing 3’ OH This end has a 3’ OH Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Circular bacterial chromosomes have one origin Eukaryotic chromosomes have hundreds or even thousands of replication origins Close-up of a replication fork Origin of replication Parental (template) strand 0.25 µm Daughter (new) strand Bubble Replication fork Two daughter DNA molecules

Overall direction of replication Synthesis of leading and lagging strands during DNA replication Parental DNA DNA pol Ill elongates DNA strands only in the 5 3 direction. 1 Okazaki fragments DNA pol III Template strand Lagging strand 3 2 DNA ligase Overall direction of replication One new strand, the leading strand, can elongate continuously 5 3 as the replication fork progresses. The other new strand, the lagging strand must grow in an overall 3 5 direction by addition of short segments, Okazaki fragments, that grow 5 3 (numbered here in the order they were made). DNA ligase joins Okazaki fragments by forming a bond between their free ends. This results in a continuous strand. 4 Figure 16.14 3 5 Leading strand

Model for the “replication machine,” or replisome

Model for the “replication machine,” or replisome Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Detail of the lagging strand Primase joins RNA nucleotides into a primer. 1 Overall direction of replication 3 5 1 2 Template strand RNA primer Okazaki fragment Figure 16.15 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 2 After reaching the next RNA primer (not shown), DNA pol III falls off. 3 After the second fragment is primed. DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 4 DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2. 5 DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. 6 The lagging strand in this region is now complete. 7

First look at leading strand: Helicase unwinds a bit of DNA Primase adds an RNA primer Polymerase adds complementary nucleotides to 3’ OH and slides down the molecule, continuing to extend the sugar-phosphate chain. (E. coli has about 40,000 turns in the circular chromosome-- each of them must be unwound to separate the DNA strands)

Now look at lagging strand Can’t form continuous molecule, since synthesis only happens 5’ to 3’. SO, need many primers and lots of short fragments Primase adds an RNA primer and Polymerase adds nucleotides until it reaches the previous primer Another polymerase removes RNA primer on adjacent stand and adds dNTPs Ligase connects the fragments

Table 16.1 Bacterial DNA replication proteins and their functions

Try this at home: Draw a replication bubble, label the bases on the template, primer, and newly synthesized DNA using the shorthand DNA notation. Convince yourself that synthesis must go 5’ to 3’ and that replication must be semi-discontinuous.