GIO: L3 02-03 DNA REPLICATION + REPAIR

WHAT IS THE DOUBLE HELIX?

• DNA IS DOUBLE-STRANDED

• THE SUGAR-PHOSPHATE BACKBONE COVALENTLY LINKS THE DEOXYRIBONUCLEOSIDES

• HYDROGEN BONDS MEDIATE BASE PAIRING

• A ONLY PAIRS WITH T

• G ONLY PAIRS WITH C

• THE COMPLEMENTARY STRANDS ARE ANTI-PARALLEL

• “IT HAS NOT ESCAPED OUR NOTICE THAT THE SPECIFIC PIRING, WE HAVE POSTULATED IMMEDIATELY SUGGESTS A POSSIBLE COPYING MECHANISM FOR THE GENETIC MATERIAL” WATSON+CRICK 1953

WHAT IS REPLICATION: DNA—>DNA

• EACH STRAND SERVES AS A TEMPLATE

• REPLICATION IS SEMI-CONSERVATIVE

• BECAUSE A ONLY PAIRS WITH T, AND G ONLY PAIRS WITH C, DNA CAN ACT AS A TEMPLATE FOR ITS OWN REPLICATION

WHAT ARE THE 3 MODELS FOR DNA REPLICATION?

• SEMICONSERVATIVE (PREDICTED BY WATSON+CRICK)

• DISPERSIVE

• CONSERVATIVE

WHAT WAS THE EXPERIMENT TO DETERMINE THE MECHANISM OF DNA REPLICATION?

LOOK AT IMAGE

WHAT WAS THE MESELSON-STAHL EXPERIMENT?

• THESE RESULTS CONFIRMED THE SEMI-CONSERVATIVE MODEL

• TO DISTINGUISH BETWEEN THE DISTRIBUTIVE AND SEMI-CONSERVATIVE MODELS, THE RESEARCHERS DENATURED THE DNA INTO SINGLE STRANDS AND FOUND THAT BOTH HEAVY AND LIGHT SINGLE STRANDS WERE OBSERVED

• THE OBSERVATION OF DNA MOLECULES OF INTERMEDIATE WEIGHT RULED OUT THE CONSERVATIVE MODEL

WHAT IS DNA REPLICATION?

• IT IS SEMI-CONSERVATIVE

WHERE DOES DNA SYNTHESIS BEGIN (FLASHCARD 1)?

• IT BEGINS AT REPLICATION ORIGINS

• INITIATOR PROTEINS BIND SPECIFIC SEQUENCES AT ORIGINS AND BREAK HYDROGEN BONDS

• IN BACTERIA AND YEAST, ORIGINS ARE ~100 BP AND ARE AT-RICH

  • AT BPS HAVE 2 HYDROGEN BONDS

  • GC BPS HAVE 3 HYDROGEN BONDS

• NUMBER OF ORIGINS PER GENOME

  • BACTERIA=ONE

  • HYMANS=~10000

WHERE DOES DNA SYNTHESIS BEGIN (FLASHCARD 2)?

• AT REPLICATION ORGINS AND IS BI-DIRECTIONAL

  • REPLICATION FORKS MOVE IN OPPOSITE DIRECTIONS

• RATE OF REPLICATION FORK MOVEMENT:

  • BACTERIA~1000 BP PER SECOND

  • HUMANS ~100 BP PER SECOND

DNA SYNTHESIS IS ALWAYS…

5’—>3’!

DNA SYNTHESIS IS PERFORMED BY WHAT? (PART 1)

• DNA POLYMERASES!!!

• REQUIRES:

  • TEMPLATE STRAND TO COPY

  • PRIMER STRAND WITH 3’ OH

  • dNTP SUBSTRATES

• CATALYZES:

  • NUCLEOPHILIC ATTACK BY 3’ OH

  • PHOSPHODIESTER BOND FORMATION

  • 5’—>3’ SYNTHESIS

DNA SYNTHESIS IS PERFORMED BY WHAT? (PART 2)

• PHOSPHODIESTER BONDS ARE FORMED BETWEEN THE 3’ OH AND THE 5’ PHOSPHATE OF THE INCOMING NUCLEOTIDE

• INCOMING NUCLEOTIDES ARE DEOXYRIBONUCLEOSIDE TRIPHOSPHATES, WHICH PROVIDE ENERGY FOR THE REACTION

• DNA POLYMERASE IS THE CORE ENZYME IN THE REPLICATION MACHINERY

DNA SYNTHESIS IS PERFORMED BY WHAT? (PART 3)

• PHOSPHODIESTER BONDS ARE FORMED BETWEEN THE 3’ OH AND THE 5’ PHOSPHATE OF THE INCOMING NUCLEOTIDE

• INCOMING NUCLEOTIDES ARE DEOXYRIBONUCLEOSIDE TRIPHOSPHATES, WHICH PROVIDE ENERGY FOR THE REACTION

• DNA POLYMERASE IS THE CORE ENZYME IN THE REPLICATION MACHINERY

WHAT DO THE NEW STRANDS HAVE?

• THEY HAVE OPPOSITE POLARITIES AT EACH REPLICATION FORK

DNA IS SYNTHESIZED BOTH…

• CONTINUOUSLY AND DISCONTINUOUSLY AT EACH REPLICATION FORK!

• THE LEADING STRAND IS CONTINUOUSLY SYNTHESIZED

• LAGGING STRAND SYNTHESIS IS DISCONTINUOUS

• OKAZAKI FRAGMENTS ARE THE SHORT SEGMENTS OF DNA THAT ARE LATER JOINED TOGETHER

DNA POLYMERASE IS…

• VERY ACCURATE!!!

• ABOUT 1 ERROR IN EVERY 10⁷ NUCLEOTIDES ADDED

• INCORRECT BASE PAIRING LEADS TO MUTATIONS

• DNA POLYMERASE ONLY ADDS NUCLEOTIDES WHEN BASE PAIRING IS CORRECT

• DNA POLYMERASE HAD PROOF-READING ACTIVITY

  • PROOF-READING IS CARRIED OUT BY THE 3’—→5’ EXONUCLEASE ACTIVITY OF THE ENZYME AND IS PERFORMED AT A SEPARATE CATALYTIC SITE

RNA PRIMERS ARE …

• REQUIRED TO BEGIN DNA REPLICATION

• DNA POLYMERASES REQUIRE:

  • TEMPLATE STRAND TO COPY

  • PRIMER STRAND WITH 3’ OH

• PRIMASE SYNTHESIZES A SHORT 10 NT PRIMER

  • PRIMASE IN AN RNA POLYMERASE

  • LEADING STRAND: ONLY ONE PRIMER

  • LAGGING STRAND: MANY PRIMERS

LAGGING STRAND SYNTHESIS…

• REQUIRES MULTIPLE ENZYMES!

• ONCE DNA POLYMERASE COMPLETES A SEGMENT:

  • NUCLEASE REMOVES THE RNA PRIMER

  • REPAIR DNA POLYMERASE REPLACES THE RNA WITH DNA

  • DNA LIGASE SEALS THE NICK

WHAT SEALS THE NICK?

• DNA LIGASE!

• DNA LIGASE ATTACHES A MOLECULE OF ATP TO THE 5’ PHOSPHATE AT THE NICK

• A NUCLEOPHILIC ATTACK BY THE 3’ HYDROXYL FORMS A NEW PHOSPHODIESTER BOND WITH THE RELEASE OF AMP

WHAT IS THE REPLICATION MACHINE?

• THE SLIDING CLAMP KEEPS THE POLYMERASE LOADED ON THE DNA

• THE DNA HELICASE USES THE ENERGY OF ATP TO UNWIND DNA

• SINGLE-STRAND DNA BINDING PROTEINS KEEP THE STRANDS APART AND ALLOW ACCESS FOR PRIMASE AND POLYMERASE

• THE CLAMP LOADER:

  • LOADS SLIDING CLAMPS ON THE LEADING AND LAGGING STRANDS

  • TETHERS THE DNA POLYMERASES TO ONE ANOTHER AT THE REPLICATION FORK

DNA TOPOISOMERASE…

• RELIEVES TENSION DUE TO UNWINDING!!!

• DNA TOPOISOMERASE CREATES SINGLE-STRAND NICKS THAT RELIEVE TORSIONAL (TWISTING) STRESS

WHAT PROTEINS ARE INVOLVED IN DNA REPLICATION?

LOOK AT IMAGE!

WHAT ARE EUKARYOTIC CHROMOSOMES?

• THEY ARE LONG AND LINEAR

• BY CONTRAST, BACTERIAL CHROMOSOMES ARE CIRCULAR, WITH A SINGLE ORIGIN

• BUT LINEAR CHROMOSOMES PRESENT A PROBLEM…

WHAT IS TELOMERASE AND THE END REPLICATION PROBLEM?

• IN HUMANS, THE TELOMERE REPEAT IS 5’-(TAAGGG)_N 3’-(ATTCCC)_N

WHAT IS DNA REPAIR: MUTATIONS?

• MUTATION: A PERMANENT CHANGE IN THE DNA SEQUENCE

• MUTATIONS CAN BE:

  • SILENT—→ NO EFFECT ON GENE FUNCTION

  • DELETERIOUS—>IMPAIRS GENE FUNCTION

  • ADVANTAGEOUS—>ENHANCES GENE FUNCTION

• MUTATIONS CAN LEAD TO:

  • GENETIC DIVERSITY

  • CANCER IN SOMATIC CELLS

  • BIRTH DEFECTS IN GERM CELLS

• MUTATIONS CAN BE CAUSED BY:

  • MISTAKES IN REPLICATION

  • DNA DAMAGE

WHAT ARE EXAMPLES OF DNA DAMAGE?

• PURINE BASES ARE SPONTANEOUSLY LOST BY DEPURINATION

• DEAMINATION IS THE SPONTANEOUS LOSS OF AMINO GROUPS FROM CYTOSINE, FORMING URACIL

• UV RADIATION FROM THE SUN CAUSES THYMINE DIMER FORMATION

WHAT IS UNREPAIRED DNA DAMAGE?

• CAN LEAD TO MUTATIONS

WHAT IS FIDELITY IN DNA REPLICATION AND WHAT DOES IT REQUIRE?

• REQUIRES BOTH PROOFREADING AND REPAIR

WHAT ARE THE THREE STEPS INVOLVED IN DNA REPAIR?

• DNA REPAIR INVOLVES 3 STEPS:

  1. EXCLUSION BY NUCLEASES

  2. NUCLEOTIDE REPLACEMENT BY REPAIR DNA POLYMERASE (THIS IS THE SAME ENZYME THAT REPLACES THE RNA PRIMERS)

  3. NICK REPAIR BY DNA LIGASE (THIS IS THE SAME ENZYME THAT SEALS THE NICK DURING DNA REPLICATION)

ERRORS MADE DURING DNA REPLICATION…

• MUST BE CORRECTED TO AVOID MUTATIONS!

MISMATCH REPAIR ELIMINATES…

• REPLICATION ERRORS AND RESTORES THE ORIGINAL DNA SEQUENCE!

• MISMATCH REPAIR FIXES MOST ERRORS MADE BY THE REPLICATION MACHINERY

  • REDUCES OVERALL ERROR RATE TO 1 IN 10⁹ BASES

  • CELLS HAVE STRATEGIES TO DETERMINE WHICH STRAND IS THE NEWLY SYNTHESIZED STRAND

  • CELLS WITH DEFECTIVE MISMATCH REPAIR SYSTEMS ARE CANCER-PRONE

WHAT IS REPAIR OF DOUBLE-STRAND BREAKS…

• DOUBLE-STRANDED BREAKS ARE DANGEROUS BECAUSE THEY CAN LEAD TO CHROMOSOMAL FRAGMENTATION AND REARRANGEMENTS

• THE CELL HAS TWO REPAIR OPTIONS:

  1. NON-HOMOLOGOUS END JOINING REPAIRS BROKEN ENDS AND REJOINS THEM BY LIGATION

  2. HOMOLOGOUS RECOMBINATION

MANY DISEASES ARE CAUSED BY…

• SINGLE NUCLEOTIDE CHANGES!

• DNA DAMAGE CAN OCCUR IN:

  • SOMATIC CELLS→ SPORADIC DISEASE, CANCER

  • GERM CELLS—> GENETIC DISEASES, INHERITED IN ALL CELLS

• EXAMPLE: CANCER INCIDENCE INCREASES DRAMATICALLY WITH AGE. WHY?

  • CELLS ARE CONTINUALLY EXPERIENCING ACCIDENTAL CHANGES TO THEIR DNA-WHICH ACCUMULATE AND ARE PASSED ON WHEN THE MUTATED CELLS DIVIDE