Warm-up: Identify the components of the following parts of DNA : A nucleotide DNA Backbone DNA ladder steps What do 3’ and 5’ represent in a DNA molecule?

Modified from a PowerPoint obtained from Mrs. Boyd, WHS High School DNA Replication: How does DNA make a copy of itself? 2007-2008 Modified from a PowerPoint obtained from Mrs. Boyd, WHS High School

A. Directionality of DNA Putting the DNA backbone together a. Numbered carbons nucleotide PO4 N base 5 CH2 O This will be IMPORTANT!! 4 1 ribose 3 2 OH

Sounds trivial, but… this will be IMPORTANT!! 1. The DNA backbone 5 PO4 b. refer to the 3 and 5 ends of the DNA the last trailing carbon base CH2 5 O 4 1 C 3 2 O –O P O Sounds trivial, but… this will be IMPORTANT!! O base CH2 5 O 4 1 3 2 OH 3

QUICK CHECK: nucleotide PO4 N base 5 CH2 O 4 1 ribose 3 2 OH If DNA grows from 5’ to 3’, the next nucleotide will be added to the: A. 3’ end B. 5’ end nucleotide PO4 N base 5 CH2 O 4 1 ribose 3 2 OH

c. Anti-parallel strands Nucleotides in DNA backbone are bonded from phosphate to sugar between 3 & 5 carbons DNA molecule has “direction” complementary strand runs in opposite direction 5 3 3 5

B. Bonding in DNA hydrogen bonds 5 3 covalent bonds 3 5

QUICK CHECK: The DNA backbone is made up of _____ and _____. A. nitrogen bases and deoxyribose B. deoxyribose and a phosphate group C. nitrogen base and a phosphate group D. nitrogen base and amino acid

C. Base pairing in DNA Purines Pyrimidines Pairing Rules adenine (A) guanine (G) Pyrimidines thymine (T) cytosine (C) Pairing Rules A : T 2 bonds C : G 3 bonds

QUICK CHECK: The DNA double helix has a uniform diameter because ____, which have two rings, always pair with ____, which have one ring. A. pyrimidines, purines B. purines, pyrimidines

D. Copying DNA Replication of DNA base pairing allows each strand to serve as a template for a new strand new strand is 1/2 parent template & 1/2 new DNA

2. Models of DNA Replication a. 3 Possible Models: conservative semiconservative dispersive

E. Mechanisms of DNA Replication Let’s meet the team… Large team of enzymes coordinates replication Enzymes more than a dozen enzymes & other proteins participate in DNA replication

single-stranded binding proteins 1. Replication: 1st step a. Unwind DNA helicase enzyme unwinds part of DNA helix stabilized by single-stranded binding proteins helicase single-stranded binding proteins replication fork

Where’s the ENERGY for the bonding! We’re missing something! 2. Replication: 2nd step a. Build daughter DNA strand add new complementary bases DNA polymerase III Where’s the ENERGY for the bonding! But… We’re missing something! What? DNA Polymerase III

3. Energy of Replication ATP TTP CTP GTP AMP ADP GMP TMP CMP Where does energy for bonding usually come from? We come with our own energy! energy You remember ATP! Are there other ways to get energy out of it? energy Are there other energy nucleotides? You bet! And we leave behind a nucleotide! ATP TTP CTP GTP AMP ADP GMP TMP CMP modified nucleotide

QUICK CHECK: What kind of chemical bond is found between paired bases of the DNA double helix? A. H - bond B. covalent C. ionic

4. Direction of Replication 5 3 energy DNA Polymerase III a. Adding bases can only add nucleotides to 3 end of a growing DNA strand need a “starter” nucleotide to bond to strand only grows 53 energy DNA Polymerase III DNA Polymerase III energy DNA Polymerase III The energy rules the process. energy B.Y.O. ENERGY! The energy rules the process 3 5

need “primer” bases to add on to 5 3 5 3 need “primer” bases to add on to energy no energy to bond  energy energy energy energy ligase energy energy 3 5 3 5

b. Leading & Lagging strands Okazaki b. Leading & Lagging strands Limits of DNA polymerase III can only build onto 3 end of an existing DNA strand  5 Okazaki fragments 5 5 3 5 3 5 3 ligase Lagging strand 3 growing replication fork 3 5 Leading strand  3 5 Lagging strand Okazaki fragments joined by ligase 3 DNA polymerase III Leading strand continuous synthesis

Why does the lagging strand have to grow in fragments? QUICK CHECK: Why does the lagging strand have to grow in fragments? A. because DNA can only grow from 5’ to 3’ B. because DNA can only grow from 3’ to 5’ C. because DNA polymerase can only read the template from 5’ to 3’

5. Replication fork / Replication bubble 5 3 3 5 DNA polymerase III leading strand 5 3 5 3 5 5 3 lagging strand 5 3 5 3 5 3 5 lagging strand leading strand growing replication fork growing replication fork 5 leading strand lagging strand 3 5 5 5

a. Starting DNA synthesis: RNA primers 5 5 3 5 3 5 3 3 growing replication fork 5 3 primase 5 DNA polymerase III RNA RNA primer built by primase serves as starter sequence for DNA polymerase III 3

b. Replacing RNA primers with DNA DNA polymerase I removes sections of RNA primer and replaces with DNA nucleotides DNA polymerase I 5 3 ligase 3 5 growing replication fork 3 5 RNA 5 3 But DNA polymerase I still can only build onto 3 end of an existing DNA strand

direction of replication 5. Replication fork DNA polymerase III lagging strand DNA polymerase I 3’ primase Okazaki fragments 5’ 5’ ligase SSB 3’ 5’ 3’ helicase DNA polymerase III 5’ leading strand 3’ direction of replication SSB = single-stranded binding proteins

DNA polymerase III enzyme 6. DNA polymerases Roger Kornberg DNA polymerase III 1000 bases/second! main DNA builder DNA polymerase I 20 bases/second editing, repair & primer removal Arthur Kornberg DNA polymerase III enzyme In 1953, Kornberg was appointed head of the Department of Microbiology in the Washington University School of Medicine in St. Louis. It was here that he isolated DNA polymerase I and showed that life (DNA) can be made in a test tube. In 1959, Kornberg shared the Nobel Prize for Physiology or Medicine with Severo Ochoa — Kornberg for the enzymatic synthesis of DNA, Ochoa for the enzymatic synthesis of RNA.

7. Editing & proofreading DNA a. 1000 bases/second = lots of typos! b. DNA polymerase I proofreads & corrects typos repairs mismatched bases removes abnormal bases repairs damage throughout life Nucleases: DNA cutting enzymes that help repair damage reduces error rate from 1 in 10,000 to 1 in 100 million bases

What does it really look like? 1 2 3 4

Learning Log: What does anti-parallel mean? 2. What kind of chemical bond is found between paired bases of the DNA double helix? 3. What does it mean to say that the replication of DNA is semi-conservative?