DNA Replication and Repair
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Overview of DNA Replication SWYK CNs – 1, 2, 30 Explain how specific base pairing enables existing DNA strands to accurately replicate themselves.
• genetic information is passed on DNA to the next generation Replication • semi-conservative T A T T A T A A A T A T G C G G C C G C C G C G A T A A T A T T A T A T T T A T T A A A T A T A G C C C G C G G C G C G Each daughter Parent molecule Parental Each parental DNA molecule with two strands strand is a consists of one complementary parental and one separate template molecules new strand
Overview of replication Initiation • DNA is unwound and stabilized • Origins of replication: Replication bubble and replication fork Priming • RNA primers bind to sections of the DNA and initiate synthesis Elongation • Leading strand (5’ 3’) synthesized continuously • Lagging strand synthesized discontinuously then fragments are joined • RNA primer replaced by DNA Proofreading • Mismatch repair by DNA polymerase • Excision repair by nucleases
Review of DNA structure • double helix • each strand has a 5’ phosphate end and a 3’ hydroxyl end • strands run antiparallel to each other • A-T pairs (2 H-bonds), G-C pairs (3 H-bonds)
SWYK CNs – 3, 4, 29 1. Sketch and label origins of replication: Replication bubble and replication fork 2. Describe the roles of the following proteins: - helicase - single-strand binding proteins - topoisomerase 3. Give two differences between prokaryotic and eukaryotic DNA replication.
• Depend on a specific AT-rich DNA sequence STEP 1 – Prokaryotes – one site – Eukaryotes – multiple sites Initiation at origins • Replication bubble of replication • Replication fork separation sites on DNA strands • Proceeds in two directions from point of origin
The proteins of initiation and priming
SWYK CNs – 5, 6, 27 4. What is the role of primase? 5. Why do we need RNA primers in replication? 6. What is the leading strand? How is it different from the lagging strand in terms of the priming step?
STEP 2 Priming initiation of DNA synthesis by RNA RNA primers bind to unwound sections through the action of primase – leading strand – only 1 primer – lagging strand – multiple primers – replaced by DNA later
SWYK CNs – 7, 25, 26 7. Describe the roles of the following proteins: DNA polymerase (I/III) DNA ligase 8. Why is the leading strand synthesized continuously whereas the lagging strand is synthesized discontinuously? 9. Draw parental DNA being replicated. Label the following: parental DNA, leading strand, lagging strand, 3’ and 5’ ends, Okazaki fragments.
STEP 3 Elongation of a new DNA strand lengthening in the 5’ 3’ direction DNA polymerase III can only add nucleotides to the 3’ hydroxyl end Leading strand - DNA pol III – adds nucleotides towards the replication fork; - DNA pol I - replaces RNA with DNA Lagging strand - DNA pol III - adds Okazaki fragments to free 3’ end away from replication fork - DNA pol I - replaces RNA with DNA - DNA ligase – joins Okazaki fragments to create a continuous strand
SWYK CNs – 10, 11, 24 10. How does DNA polymerase III function in mismatch repair? 11. How do nuclease, DNA pol III, and ligase function in excision repair? 12. What is the problem with telomeres at the 5' end? How is this solved in eukaryotic cells? You may use a diagram and labels for this.
STEP 4 Proofreading correcting errors in replication Mismatch repair • DNA pol III – proofreads nucleotides against the template strand Excision repair • nuclease – cuts damaged segment • DNA pol III and ligase – fill the gap left Telomeres at 5’ ends of lagging strands • no genes, only 100 – 1000 TTAGGG sequences to protect genes • telomerase catalyzes lengthening of telomeres
Gene Expression From gene to protein Transcription and Translation
GENE EXPRESSION Genes code for polypeptide chains or for RNA molecules TRANSCRIPTION TRANSLATION • DNA-directed RNA • mRNA-directed synthesis polypeptide synthesis • produces mRNA • occurs on ribosomes • Prokaryotes – mRNA translated immediately • Eukaryotes – pre-mRNA processed before leaving the nucleus as mRNA
SWYK CNs – 12, 13, 14, 22, 23 15. What is the role of RNA polymerase II in initiating transcription? 16. Assuming that the DNA template strand has the sequence 3 ’ A T A T T T T A C G C G C C A 5’, draw a) Nontemplate/coding/sense strand b) RNA strand
Transcription INITIATION ELONGATION TERMINATION
STEP 1 – Initiation Occurs at a promoter Transcription factors bind to the TATA box on the DNA. RNA polymerase II and other transcription factors bind to promoter. DNA strands unwind. Polymerase initiates RNA synthesis at the start point.
STEP 2 – Elongation mRNA transcript lengthens New RNA DNA RNA Polymerase strand strands re- polymerase adds peels away form a unwinds nucleotides from double DNA. to 3’ end. template. helix.
Nontemplate Template • Coding • Noncoding • Sense • Antisense
STEP 3 – Termination mRNA transcript released Polymerase transcribes Polymerase pre-mRNA is polyadenylation detaches from released signal sequence DNA. (AAUAAA)
SWYK CNs – 15, 21 17. mRNA in eukaryotes undergoes RNA processing. Draw, label, and explain the significance of the following structures a. 5 ’ cap b. poly-A tail c. spliceosome
RNA processing Protect mRNA from Facilitate export of degradation by alteration of pre- mRNA from nucleus enzymes mRNA Help ribosomes attach to 5’ end of RNA 5’ cap Poly-A tail RNA splicing Modified G added 50-250 A added to 3’ end to 5’ end
• Introns – intervening sequences – RNA processing noncoding segments on pre-mRNA RNA splicing – May regulate gene activity – Enable genes to give rise to two or more different polypeptides – Facilitate evolution through exon shuffling • Exons – expressed sequences on pre- mRNA • Signal for splicing is a short sequence at the ends of introns
• small nuclear ribonucleoproteins (snRNPs) with snRNA recognize splice sites • snRNPs + proteins spliceosome – release introns – join together exons that flank introns
SWYK CNs – 16, 17, 18, 19, 20 Initiator tRNA base with the anticodon (17. _________) pairs with the start codon (18. _________ ) 19. Draw and label the initiator tRNA, mRNA, start codon, and large ribosomal subunits with EPA sites during initiation of translation. Ribosome reaches STOP codons (20.) ___________, _________, _________)
Translation Overview • mRNA moves through ribosome • codons are translated into amino acids • tRNA molecules: anticodon and amino acid ends • amino acids added to a growing polypeptide chain • rRNA molecules + proteins ribosomes
tRNA structure
SWYK CNs – 8, 9 21. What is wobble? What is its significance? 22. What is the genetic code? Why is it described as redundant but not ambiguous? 23. Given a prokaryotic DNA template with the following sequence, write the sequence of the mRNA transcript formed and the polypeptide synthesized: 3’ T A T A A T C T A C A C A T T G C C G T A C T A A A T A 5’,
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Inosine
Ribosome structure
Building a polypeptide Initiation Translation Termination
Step 1 - Initiation Initiator Initiator Large R Small R tRNA base tRNA in the subunit binds subunit pairs with P site, A to complete binds to the initiation start codon site is mRNA complex ( AUG ) empty
Step 2 Elongation
Step 3 – Termination Release factor A site Ribosome cleaves bond Two receives a reaches STOP between ribosomal codon ( UAG, release tRNA and the subunits UAA, UGA ) last amino disassemble factor acid
Given the following sequence on a template DNA strand 3’ AAA TAT TTT CCG TAC GGA TAG ACA CCG AAA ATC CGG GCA 5’ • What is the sequence on the non-template strand? 5’ TTT ATA AAA GGC ATG CCT ATC TGT GGC TTT TAG GCC CGT 3’ • What is the mRNA sequence transcribed (assuming transcription right occurs after the TATA sequence)? 5’ AAA GGC AUG CCU AUC UGU GGC UUU UAG GCC CGU 3’ • What is the STOP codon? UAG • What is the anticodon attached to the tRNA that corresponds to the STOP codon? There is no tRNA that corresponds to the STOP codon. Release factors take their place. • What is the amino acid sequence in the polypeptide product? Met – Pro – Ile – Cys – Gly - Phe
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