Unit 3 Synthesis of DNA

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1

Origin of Replication

Single site in prokaryotes. Multiple sites along genomic DNA in eukaryotes- usually at AT rich sites.

Replication is bidirectional.

2

Pre-priming Complex

DNA-A: binds and melts dsDNA; DNA helicase: unwinds dbl helix; ssDNA-binding proteins: keep strands separate & protect from nucleases. Moves along template 3'-5'

3

Solving Supercoiling

unwinding DNA forms positive supercoils ahead of replication fork; DNA topoisomerase removes supercoils & facilitates transcription (nicks one of two strands)

4

Direction of DNA Replication

Leading strand: copied in direction of replication fork, synth. continuously; Lagging strand copied away from replication fork, Okazaki Fragments (small fragments of DNA) discontinuously

5

Initiation and Elongation (Prokaryotic)

DNA polymerase require RNA primer to initiate synth.- accomplished with Primase. DNA pol III initiates replication using RNA primer, and elongation (highly processive), and proofreading DURING replication

6

Replacement of RNA primers (Prokaryotic)

DNA pol I: locates space between DNA & RNA, replaces RNA w/ DNA; DNA fragments linked by DNA ligase

7

Pol I; Pol II; Pol III Table (Prokaryotic)

Pol I: 5'-3' & 3'-5' exonuclease actvty, remove primer in conjunction w/ RNAse H, DNA repair, fill gap after removal of RNA primer; Pol II: 3'-5' exonuclease actvty, DNA repair; Pol III 3'-5' exonuclease actvty, Replication-synth of DNA

8

DNA Ligase

forms phosphodiester bond between 3' OH & 5' phosphate of nucleotides w/ ATP, DNA repair actvty, used in recombinant DNA tech

9

Eukaryotic DNA Replication

Multiple Ori sites, RNA primers removed by RNAse H, DNA replication and cell division coordinated with cell cycle

10

Cell Cycle (Eukaryotic)

G1- precedes replication, S- replication/ synthesis phase, G2- mitosis, G0- cells that stop dividing

11

Eukaryotic DNA Plymerases

No Proof Reading- *Pol alpha: contains DNA primase and initiates DNA synthesis, *Pol beta: Repair;

Proof Reading- *Pol gamma: replicates mito DNA, *Pol delta: elongates leading and lagging strands, *Pol epsilon: repair

12

Shortening of DNA with Replication

No way to fill gap after removal of primer from extreme 5' end- may cause aging

13

Telomeres

Region of highly repetitive DNA at end of chromosome (TTAGGG)- many Kbp in humans, *buffer consumed during cell division, *protect ends from nucleases, *fixed number of divisions, loss of DNA leads to *senescence & cell death

14

Telomerase

active in germ, stem, and cancer cells- elongates telomeres. Reactivated by physical exercise (aorta & mononuclear cells), inc. vit. D (in leukocytes), TA-65 telomerase activator; *Ribonucleoprotein complex: proteins & RNA, RNA-dependent DNA pol; extends 3' end of DNA

15

DNA Repair Rate

up to 500K modification events each day per cell

16

Action of Mutagens

Indirect: *X-rays form free radicals from H2O; Direct: *cigarette smoke contains carcinogen benzo(a)pyrene- when oxidized forms adducts with guanine residues, *UV leads to dimer formation between thymines on DNA

17

Xeroderma pigmentosum

autosomal recessive, defect in nucleotide excision repair (NER) system, cells cannot repair damaged DNA...skin cancer

18

Repair Mechanisms

E.coli- photoactivating enzymes for Thymine dimers; Nucleotide excision repair: *Endonuclease removes distorted region; Base excision repair: *DNA glycosylases cleave N-glycoside bond, *AP endonuclease cleaves sugar-phosphate strand

19

Which Strand to Repair?

Prokaryotic: Parental DNA is methylated. Not clearly understood in humans.

20

Reverse Transcriptase Inhibitors

*didanosine (ddl): dideoxynucleoside converted to ddlTP in cells and *terminates elongation when incorporated by reverse transcriptase.