The Molecular Basis of Inheritance DNA-the Genetic Material DNA-Replication and Repair

DNA-the Genetic Material Protein was first thought to be the genetic stuff. Why? Evidence for DNA came from failed experiments by Griffith

Searching for Genetic Material, I Mendel: modes of heredity in pea plants Morgan: genes located on chromosomes Griffith: bacterial work; transformation: change in genotype and phenotype due to assimilation of external substance (DNA) by a cell Avery: transformation agent was DNA

Searching for Genetic Material, II Hershey and Chase √ bacteriophages (phages) √ DNA, not protein, is the hereditary material √ Expt: sulfur(S) is in protein, phosphorus (P) is in DNA; only P was found in host cell

Further evidence Hershey and Chase- bacteriaphage experiments Chargaff’s Rules Franklin-X-ray crystalography

Watson and Crick explained Chargaff’s rules of base pairing Presented model of DNA in1953 explained Franklins repeating measurements explained antiparallel nature of DNA

DNA Structure Double helix antiparallel strands composed of nucleotides nucleotides made of deoxyribose, nitrogenous base and a phosphate group

Base pairing Purine with a pyrimidine maintains 2.0 nm molecular diameter Purines: adenine and guanine pyrimidines: thymine and cytosine adenine-thymine cytosine-guanine held together by H-bonds

DNA Replication begins at sites called Origins of Replication DNA unravels to form two replication forks Semi-conservative DNA polymerase adds new nucleoside triphosphates only in 5’ to 3’ direction exergonic hydrolysis of phyrophosphates drives DNA synthesis

C Nucleotide Different Ways to Represent the DNA Double Helix Slide number: 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. P P P P P P P P P P P P A C G T C G C G A C G T AB T G A C T G A C T G A C A G C T G A C T G

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Nucleotide Different Ways to Represent the DNA Double Helix Slide number: 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. P A

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DNA Replication: a closer look Origin of replication (“bubbles”): beginning of replication Replication fork: ‘Y’-shaped region where new strands of DNA are elongating Helicase:catalyzes the untwisting of the DNA at the replication fork DNA polymerase:catalyzes the elongation of new DNA

DNA Replication, II Antiparallel nature: sugar/phosphate backbone runs in opposite directions (Crick); one strand runs 5’ to 3’, while the other runs 3’ to 5’; DNA polymerase only adds nucleotides at the free 3’ end, forming new DNA strands in the 5’ to 3’ direction only

DNA Replication, III Leading strand: synthesis toward the replication fork (only in a 5’ to 3’ direction from the 3’ to 5’ master strand) Lagging strand: synthesis away from the replication fork (Okazaki fragments); joined by DNA ligase (must wait for 3’ end to open; again in a 5’ to 3’ direction) Initiation: Primer (short RNA sequence~w/primase enzyme), begins the replication process

The Time Factor discontinuous and continuous syntheses Limited time for replication role of Okazaki fragments role of ligases

12.15 DNA Replication Takes Many Steps Slide number: 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Enzymes in DNA replication Helicase unwinds parental double helix Binding proteins stabilize separate strands DNA polymerase binds nucleotides to form new strands Primase adds short primer to template strand Ligase joins Okazaki fragments and seals other nicks in sugar- phosphate backbone Helicase binds to origin and separates strands. 1 Binding proteins prevent single strands from rejoining. 2 Primase makes a short stretch of RNA on the DNA template. 3 DNA polymerase adds DNA nucleotides to the RNA primer. 4 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 Leading (continuous) strand synthesis continues in a 5’ to 3’ direction. 6 Discontinuous synthesis produces Okazaki fragments on the 5’ to 3’ template. 7 Enzymes remove RNA primers. Ligase seals sugar-phosphate backbone. 8 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Overall direction of replication 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Okazaki fragment

1 5’ 3’ 5’ 3’ DNA Replication Takes Many Steps Slide number: 2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Helicase binds to origin and separates strands. 1

12.15 DNA Replication Takes Many Steps Slide number: 3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2 5’ 3’ 5’ 3’ Binding proteins prevent single strands from rejoining. 2 Helicase binds to origin and separates strands. 1

12.15 DNA Replication Takes Many Steps Slide number: 4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 3’ 5’ 3’ 5’ 3’ Primase makes a short stretch of RNA on the DNA template. 3 Helicase binds to origin and separates strands. 1 Binding proteins prevent single strands from rejoining. 2

12.15 DNA Replication Takes Many Steps Slide number: 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 3’ 5’ 3’ 5’ 3’ 5’ 3’ Primase makes a short stretch of RNA on the DNA template. 3 Helicase binds to origin and separates strands. 1 Binding proteins prevent single strands from rejoining. 2

Overall direction of replication DNA Replication Takes Many Steps Slide number: 6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 4 5’ 3’ 5’ 3’ 5’ 3’ 5’ DNA polymerase adds DNA nucleotides to the RNA primer. 4 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 Binding proteins prevent single strands from rejoining. 2

5’ 3’ Overall direction of replication DNA Replication Takes Many Steps Slide number: 7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 4 5’ 3’ 5’ 3’ 5’ DNA polymerase adds DNA nucleotides to the RNA primer. 4 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 Binding proteins prevent single strands from rejoining. 2

4 5’ 3’ 5’ DNA Replication Takes Many Steps Slide number: 8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Overall direction of replication 3’ 5’ 3’ DNA polymerase adds DNA nucleotides to the RNA primer. 4 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 Binding proteins prevent single strands from rejoining. 2

5’ 4 3’ DNA Replication Takes Many Steps Slide number: 9 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Overall direction of replication 5’ 3’ 5’ 3’ DNA polymerase adds DNA nucleotides to the RNA primer. 4 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 Binding proteins prevent single strands from rejoining. 2

5 5’ 3’ 5’ DNA Replication Takes Many Steps Slide number: 10 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Overall direction of replication 5’ 3’ 5’ 3’ DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 DNA polymerase adds DNA nucleotides to the RNA primer. 4 Binding proteins prevent single strands from rejoining. 2

5’ 6 3’ DNA Replication Takes Many Steps Slide number: 11 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5’ 3’ 5’ 3’ Overall direction of replication Leading (continuous) strand synthesis continues in a 5’ to 3’ direction. 6 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 DNA polymerase adds DNA nucleotides to the RNA primer. 4 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 Binding proteins prevent single strands from rejoining. 2

5’ 3’ 7 5’ DNA Replication Takes Many Steps Slide number: 12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Overall direction of replication Okazaki fragment 5’ 3’ 5’ 3’ Discontinuous synthesis produces Okazaki fragments on the 5’ to 3’ template. 7 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 DNA polymerase adds DNA nucleotides to the RNA primer. 4 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 Leading (continuous) strand synthesis continues in a 5’ to 3’ direction. 6 Binding proteins prevent single strands from rejoining. 2

5’ 3’ 7 5’ DNA Replication Takes Many Steps Slide number: 13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Overall direction of replication Okazaki fragment 5’ 3’ 5’ 3’ 5’ Discontinuous synthesis produces Okazaki fragments on the 5’ to 3’ template. 7 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 DNA polymerase adds DNA nucleotides to the RNA primer. 4 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 Leading (continuous) strand synthesis continues in a 5’ to 3’ direction. 6 Binding proteins prevent single strands from rejoining. 2 3’

5’ DNA Replication Takes Many Steps Slide number: 14 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Discontinuous synthesis produces Okazaki fragments on the 5’ to 3’ template. 7 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 DNA polymerase adds DNA nucleotides to the RNA primer. 4 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 Leading (continuous) strand synthesis continues in a 5’ to 3’ direction. 6 Binding proteins prevent single strands from rejoining. 2

5’ DNA Replication Takes Many Steps Slide number: 15 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Discontinuous synthesis produces Okazaki fragments on the 5’ to 3’ template. 7 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 DNA polymerase adds DNA nucleotides to the RNA primer. 4 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 Leading (continuous) strand synthesis continues in a 5’ to 3’ direction. 6 Binding proteins prevent single strands from rejoining. 2

12.15 DNA Replication Takes Many Steps Slide number: 16 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5’ 3’ 5’ 3’ 5’ 3’ 5’ Enzymes remove RNA primers.8 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 DNA polymerase adds DNA nucleotides to the RNA primer. 4 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 Leading (continuous) strand synthesis continues in a 5’ to 3’ direction. 6 Discontinuous synthesis produces Okazaki fragments on the 5’ to 3’ template. 7 Binding proteins prevent single strands from rejoining. 2

12.15 DNA Replication Takes Many Steps Slide number: 17 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3’ 5’ 3’ 5’ 3’ Enzymes remove RNA primers. Ligase seals sugar-phosphate backbone. 8 Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 DNA polymerase adds DNA nucleotides to the RNA primer. 4 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 Leading (continuous) strand synthesis continues in a 5’ to 3’ direction. 6 Discontinuous synthesis produces Okazaki fragments on the 5’ to 3’ template. 7 Binding proteins prevent single strands from rejoining. 2 3’

12.15 DNA Replication Takes Many Steps Slide number: 18 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Helicase binds to origin and separates strands. 1 Primase makes a short stretch of RNA on the DNA template. 3 DNA polymerase adds DNA nucleotides to the RNA primer. 4 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 Leading (continuous) strand synthesis continues in a 5’ to 3’ direction. 6 Discontinuous synthesis produces Okazaki fragments on the 5’ to 3’ template. 7 Binding proteins prevent single strands from rejoining. 2 Enzymes remove RNA primers. Ligase seals sugar-phosphate backbone. 8

Priming New DNA strands must attach to RNA RNA attached to template DNA by primase DNA polymerase removes primer as lagging strands are formed

Helicases and other proteins Helicase: enzyme that unwinds DNA at origins of replication Single-strand binding proteins: “spacers” that prevent DNA from reattaching

Errors? Replication highly accurate 1 error per 10,000 (initially) Mismatch repair- several proteins + DNA polymerase Excision repair

Telomeres Repeats of short noncoding nucleotide sequences each time DNA replicates, the sequence is shortened. Consequences?