DNA - The Code of DNA - The Code of Life Life

Chapter 16Chapter 16

Bacterial TransformationBacterial Transformation

F. Griffith & TransformationF. Griffith & Transformation• 19281928• Streptococcus pneumoniae Streptococcus pneumoniae

2 strains: R – harmless, S – pathogenic2 strains: R – harmless, S – pathogenic

• Mixed inactive S strain & active R Mixed inactive S strain & active R strain; injected into mousestrain; injected into mouse

• Mouse died, pathogenic strain in Mouse died, pathogenic strain in bloodblood

• TransformationTransformation

Avery, McCarty & MacLeodAvery, McCarty & MacLeod

• 1944 : transforming agent is DNA1944 : transforming agent is DNA• Skeptics – bacteria not complexSkeptics – bacteria not complex• More researchMore research

Viruses (bacteriophages)Viruses (bacteriophages) Viral structure & replication?Viral structure & replication?

yy

Hershey & Chase – The Blender Hershey & Chase – The Blender Experiment Experiment

• 1952: DNA is the genetic material of 1952: DNA is the genetic material of the phage T2the phage T2

• T2 phage infects T2 phage infects E. coliE. coli• Labeled Labeled protein coatprotein coat with radioactive with radioactive

S and the S and the DNA DNA with radioactive Pwith radioactive P• Phages infect Phages infect E. coliE. coli separately separately• Only P found in the bacterium Only P found in the bacterium

FindingsFindings

• Infected with radio-labeled proteins - Infected with radio-labeled proteins - radioactivity in supernatantradioactivity in supernatant

• Infected with radio-labeled DNA - Infected with radio-labeled DNA - radioactivity in pellet radioactivity in pellet

• Hershey & Chase’s conclusion?Hershey & Chase’s conclusion?

Let the race begin!Let the race begin!

• 1950s – scientific community racing 1950s – scientific community racing to find to find 3-D3-D structure of DNA structure of DNA

• Major playersMajor players James Watson & Francis Crick James Watson & Francis Crick Linus PaulingLinus Pauling Maurice Wilkins & Rosalind FranklinMaurice Wilkins & Rosalind Franklin

Puzzle PiecesPuzzle Pieces

1.1. Chargaff’s DataChargaff’s Data

2.2. Backbone Structure – single strandBackbone Structure – single strand

3.3. Franklin’s DataFranklin’s Data

Chargaff’s Rules - StructureChargaff’s Rules - Structure

• 1947 1947 • Polymer - deoxyribose sugar, Polymer - deoxyribose sugar,

phosphate grp & nitrogen-containing phosphate grp & nitrogen-containing basebase

• Bases: Bases: adenine (A), thymine (T), guanine (G), or adenine (A), thymine (T), guanine (G), or

cytosine (C)cytosine (C)

Chargaff’s RulesChargaff’s Rules

• Certain bases were always equal in Certain bases were always equal in number number # adenines approx equal to # of # adenines approx equal to # of

thymines (%T = %A)thymines (%T = %A) # guanines approx equal to # of # guanines approx equal to # of

cytosines (%G = %C)cytosines (%G = %C)

• Why is this significant?Why is this significant?

Backbone StructureBackbone Structure• Phosphate

group of one nucleotide attached to the sugar of the next

• Result is a “backbone” of phosphates and sugars, from which the bases project

R. Franklin & M. Wilkins

X-Ray Crystallography

Franklin’s DataFranklin’s Data

• Wilkins & Franklin used Wilkins & Franklin used X-ray X-ray crystallographycrystallography to study DNA to study DNA structure structure

• X-rays diffracted as they pass X-rays diffracted as they pass through aligned fibers of purified through aligned fibers of purified DNADNA

• Diffraction pattern used to deduce Diffraction pattern used to deduce 3-D shape of molecules3-D shape of molecules

What the picture tells usWhat the picture tells us

• DNA was helical in shape!DNA was helical in shape!

Putting the puzzle Putting the puzzle together…together…

• Watson & Crick – “stolen” ideas or a Watson & Crick – “stolen” ideas or a product of the times?product of the times?

• However… it was Watson who However… it was Watson who deduced the width of the helixdeduced the width of the helixand the spacing of basesand the spacing of bases

• Model building to beat PaulingModel building to beat Pauling

TRIAL AND ERRORTRIAL AND ERROR

• Watson & Crick began to work on a Watson & Crick began to work on a model of DNA with two strands, the model of DNA with two strands, the double helixdouble helix

• Wire molecular models - first tried to Wire molecular models - first tried to place the sugar-phosphate chains on place the sugar-phosphate chains on the insidethe inside

• Did not fit the X-ray measurements Did not fit the X-ray measurements and other info on chemistry of DNAand other info on chemistry of DNA

BreakthroughBreakthrough• Watson put sugar-phosphate chain Watson put sugar-phosphate chain

on the outside & nitrogen bases on on the outside & nitrogen bases on the insidethe inside

Nuts & Bolts – Chargaff’s DataNuts & Bolts – Chargaff’s Data

• Watson & Crick determined that Watson & Crick determined that chemical side groups off nitrogen chemical side groups off nitrogen bases formed H bonds, connecting bases formed H bonds, connecting strandsstrands Adenine - form 2 H bonds Adenine - form 2 H bonds onlyonly with with

thymine thymine Guanine - form 3 H bonds Guanine - form 3 H bonds onlyonly with with

cytosinecytosine Findings explained Chargaff’s rulesFindings explained Chargaff’s rules

DNA StructureDNA Structure• DNA bases:DNA bases:

Purines: adenine and guaninePurines: adenine and guanine Pyrimidines: thymine and cytosinePyrimidines: thymine and cytosine

• Nucleotides are covalently bonded Nucleotides are covalently bonded with a sugar-phosphate backbonewith a sugar-phosphate backbone

• The linkage forms a 3’,5’ The linkage forms a 3’,5’ phosphodiester linkagephosphodiester linkage

• One end of the molecule has a free One end of the molecule has a free 5’ carbon; the other has a free 3’ 5’ carbon; the other has a free 3’ carboncarbon

DNA StructureDNA Structure• Two polynucleotide chains Two polynucleotide chains

intertwined to form a double helixintertwined to form a double helix

Technical DNA DataTechnical DNA Data

• .34 nm is the distance between the .34 nm is the distance between the basesbases

• 3.4 nm repeat of nucleotides due to a 3.4 nm repeat of nucleotides due to a complete “turn” of the helixcomplete “turn” of the helix

• width of molecule is 2.0 nmwidth of molecule is 2.0 nm

Pyrimidines and PurinesPyrimidines and Purines

• PyrimidinesPyrimidines are single-ringed are single-ringed• PurinesPurines are double-ringed are double-ringed• Bonding is Bonding is complementarycomplementary

Sequence in one chain dictates Sequence in one chain dictates sequence in opposite chainsequence in opposite chain

DNA ReplicationDNA Replication• Complimentary bases act as Complimentary bases act as

templatestemplates• Bases of one strand allow for exact Bases of one strand allow for exact

duplicationduplication

DNA ReplicationDNA Replication• Origin(s) of replicationOrigin(s) of replication• ProkaryotesProkaryotes - single specific - single specific

sequence of nucleotides recognized sequence of nucleotides recognized by replic. enzymesby replic. enzymes Replication proceeds in both directionsReplication proceeds in both directions

• Eukaryotes Eukaryotes – hundreds/thousands of – hundreds/thousands of origin sites per chromorigin sites per chrom Bubble with Bubble with replication forksreplication forks at each at each

endend Bubbles elongate as DNA is replicated Bubbles elongate as DNA is replicated

and eventually fuseand eventually fuse

Bidirectional SynthesisBidirectional Synthesis• In prokaryotes, the circular DNA is In prokaryotes, the circular DNA is

opened up, and synthesis occurs in opened up, and synthesis occurs in both directionsboth directions

• In eukaryotes, the linear DNA has In eukaryotes, the linear DNA has many replication forksmany replication forks

Bidirectional SynthesisBidirectional Synthesis

DNA ReplicationDNA Replication• Proteins and enzymes work togetherProteins and enzymes work together• DNA strands must be unwound during DNA strands must be unwound during

replicationreplication DNA helicaseDNA helicase unwinds the strands unwinds the strands Single stranded binding proteins (SSB) Single stranded binding proteins (SSB)

prevent immediate reformation of the prevent immediate reformation of the double helixdouble helix

TopoisomerasesTopoisomerases break and then rejoin break and then rejoin the strands, “untying” the knots that the strands, “untying” the knots that formform

DNA Replication OrderDNA Replication Order• Always proceeds Always proceeds

in a 5’ in a 5’ 3’ 3’ directiondirection

• DNA polymeraseDNA polymerase can add only at can add only at the 3’ endthe 3’ end

• Nucleotides are Nucleotides are polymerized and polymerized and 2 phosphates are 2 phosphates are removed in the removed in the processprocess

Nucleotide SynthesisNucleotide Synthesis

• Raw nucleotides are nucleoside Raw nucleotides are nucleoside triphosphatestriphosphates N base, deoxyribose, & a triphosphate tailN base, deoxyribose, & a triphosphate tail

• Nucleotide added, last 2 phosphate Nucleotide added, last 2 phosphate grps hydrolyzed, forming grps hydrolyzed, forming pyrophosphatepyrophosphate

• Exergonic rxn drives polymerization of Exergonic rxn drives polymerization of the nucleotide to the new strandthe nucleotide to the new strand

DNA PolDNA Pol

• DNA polymerases can DNA polymerases can onlyonly add add nucleotides to the free 3’ end of a nucleotides to the free 3’ end of a growing DNA strandgrowing DNA strand

• A new DNA strand can onlyA new DNA strand can only elongate elongate in the 5’ in the 5’ 3’ direction 3’ direction

Problems? Problems?

• One parental strand is oriented 3’ One parental strand is oriented 3’ 5’ into the fork, while the other is 5’ into the fork, while the other is oriented 5’ oriented 5’ 3’ into the fork 3’ into the fork

• At fork, At fork, only oneonly one parental strand (3’ parental strand (3’ 5’ into the fork), 5’ into the fork), leading strandleading strand,, can be used by polymerases as a can be used by polymerases as a template for a template for a continuouscontinuous complementary strandcomplementary strand

Continuous & DiscontinuousContinuous & Discontinuous

Continuous & DiscontinuousContinuous & Discontinuous

• Replication is Replication is continuous on one continuous on one strand and strand and discontinuous on the discontinuous on the otherother

• Replication begins at Replication begins at replication forksreplication forks

Okazaki fragmentsOkazaki fragments• Synthesis of the leading strand is Synthesis of the leading strand is

continuouscontinuous• The The lagging strandlagging strand (discontinuous) is (discontinuous) is

synthesized in pieces called Okazaki synthesized in pieces called Okazaki fragmentsfragments

Okazaki fragmentsOkazaki fragments

• 100 - 1000 100 - 1000 nucleotides in nucleotides in lengthlength

• Initiated by a Initiated by a separate RNA separate RNA primerprimer

• Okazaki fragments Okazaki fragments are joined together are joined together by by DNA ligaseDNA ligase

RNA PrimerRNA Primer• DNA pol DNA pol cannot cannot initiateinitiate synthesis synthesis

because it can only add nucleotides because it can only add nucleotides to end of an existing chainto end of an existing chain

• Requires an RNA primerRequires an RNA primer• PrimasePrimase,, an RNA pol, links an RNA pol, links

ribonucleotides complementary to ribonucleotides complementary to the DNA template into the primerthe DNA template into the primer RNA pol can start an RNA chain from a RNA pol can start an RNA chain from a

single template strandsingle template strand

Primers & DNA LigasePrimers & DNA Ligase• Remember - Remember - leadingleading strand requires strand requires

only only one primerone primer (continuous) (continuous)• LaggingLagging strand requires formation of strand requires formation of

many new primersmany new primers as replication fork as replication fork progresses (discontinuous)progresses (discontinuous)

• After primer formed, DNA pol can After primer formed, DNA pol can add new nucleotides until it meets add new nucleotides until it meets previous Okazaki fragmentprevious Okazaki fragment

• Primers converted to DNA before Primers converted to DNA before DNA ligaseDNA ligase joins fragments together joins fragments together

Summary Summary • At the replication fork, the leading At the replication fork, the leading

strand is copied continuously into the strand is copied continuously into the fork from a single primerfork from a single primer

• Lagging strand is copied away from Lagging strand is copied away from the fork in short segments, each the fork in short segments, each requiring a new primerrequiring a new primer

ProofreadingProofreading• Mistakes do occur!Mistakes do occur!• Rate of one error per 10,000 bpRate of one error per 10,000 bp• DNA Pol “proofreads” each new DNA Pol “proofreads” each new

nucleotide against template nucleotide against template • Enzyme removes wrong nucleotide & Enzyme removes wrong nucleotide &

resumes synthesisresumes synthesis• Final error rate: 1/1 billion Final error rate: 1/1 billion

nucleotidesnucleotides

Causes of errors?Causes of errors?• Reactive chemicals, radioactive Reactive chemicals, radioactive

emissions, X-rays, uv light emissions, X-rays, uv light • Bases often undergo spontaneous Bases often undergo spontaneous

chemical changes (normal)chemical changes (normal)• Mismatched nucleotides missed by Mismatched nucleotides missed by

DNA Pol or mutations that occur after DNA Pol or mutations that occur after DNA synthesis can often be repairedDNA synthesis can often be repaired Over 130 repair enzymes identified in Over 130 repair enzymes identified in

humanshumans

Types of RepairTypes of Repair• Mismatch repair Mismatch repair - - special enzymes special enzymes

fix incorrectly paired nucleotidesfix incorrectly paired nucleotides• Nucleotide excision repairNucleotide excision repair - -

nucleasenuclease cuts out a segment of a cuts out a segment of a damaged stranddamaged strand Gap is filled in by DNA Pol & ligaseGap is filled in by DNA Pol & ligase

Completing ReplicationCompleting Replication

• Limitations of DNA Pol creates Limitations of DNA Pol creates problems for linear DNA problems for linear DNA

• No way to complete the 5’ ends of No way to complete the 5’ ends of daughter DNA strandsdaughter DNA strands

• Repeated rounds of replication Repeated rounds of replication produce shorter and shorter DNA produce shorter and shorter DNA moleculesmolecules

TelomeresTelomeres• Ends of eukaryotic chromosomes, Ends of eukaryotic chromosomes,

the the telomerestelomeres, have special , have special nucleotide sequencesnucleotide sequences Humans - this sequence is typically Humans - this sequence is typically

TTAGGG, repeated 100 - 1,000 timesTTAGGG, repeated 100 - 1,000 times

• Telomeres protect genes from Telomeres protect genes from being eroded through multiple being eroded through multiple rounds of DNA replicationrounds of DNA replication

TelomeresTelomeres• Mechanism to Mechanism to

restore shortened restore shortened telomeres:telomeres: TelomeraseTelomerase uses uses

short molecule of short molecule of RNA as a template to RNA as a template to extend the 3’ end extend the 3’ end

Room for primase & Room for primase & DNA DNA pol to extend 5’ endpol to extend 5’ end

Doesn’t repair Doesn’t repair 3’-end “overhang,”3’-end “overhang,”but lengthens but lengthens telomeretelomere

TelomeraseTelomerase• Telomerase Telomerase not present not present in most cells in most cells

of multicellular organismsof multicellular organisms• So… DNA of dividing somatic cells So… DNA of dividing somatic cells

tend to become shortertend to become shorter• Telomere length may be limiting Telomere length may be limiting

factor in the life span of certain factor in the life span of certain tissues/organismtissues/organism

• Telomerase present in Telomerase present in germ-linegerm-line cells, ( zygotes will have long cells, ( zygotes will have long telomeres)telomeres)

CancerCancer

• Active telomerase found in active Active telomerase found in active cancerous somatic cellscancerous somatic cells

• Overcomes progressive shortening Overcomes progressive shortening that would eventually lead to self-that would eventually lead to self-destruction of the cancerdestruction of the cancer