LECTURE 7

Exceptions to central dogma

reverse transcription, RNA replication, prions

Structure of DNA nucleotides

N base, C sugar (deoxyribose), phosphate group

DNA directionality

5’ phosphate end to 3’ hydroxyl end

DNA is linked by

phosphodiester bonds

DNA double helix stabilized by

H bonds and hydrophobic stacking

hydrophobic effect

produces base stacking arrangement of DNA (also involving van der Waals interactions)

One turn of DNA

10-10.5 base pairs

Adenine and thymine bonds

2 H bonds

Guanine and cytosine bonds

3 H bonds

Chargaff’s rule

A = T, C = G

Purines

Adenine and Guanine

Pyrimidines

Cytosine and thymine

Phosphodiester bond

3’ hydroxyl of one sugar group in a nucleotide, to a phosphate group on 5’ carbon of another sugar group

Glycosidic bond

N-C linkage between 9’ N of purine base or 1’ N of pyrimidine base, to 1’ C of sugar group

DNA backbone

formed by repeating deoxyribose-phosphate units linked by phosphodiester bonds

Major and minor grooves of B-DNA

expose patterns of H bond donors and accepters unique to each base pair, allowing sequence specific recognition

AAs that make the largest number of H bonds

Arg, Lys, Ser, Thr, Asn, and Gln

Intercalating agents

flat, hydrophobic molecules that insert between base pairs to distort DNA double helix by unwinding and lengthening

Steps in prokaryotic DNA synthesis

1. Separation of complimentary strands

2. Formation of replication fork

3. Priming replication

4. Direction (5’ → 3’)

5. Elongation

6. Excision of RNA primers and their replacement by DNA

7. Ligation of nick DNA

where to open double-stranded DNA

use AT-rich origin (oriC), creating an open replication bubble w/ 2 replication forks for bidirectional DNA synthesis

how to open double helix

Helicase uses ATP to unwind parental DNA, creating replication fork

how to maintain opened DNA

Single stranded DNA-binding protein (SSB)

how to start DNA replication

Primase makes short RNA primer on template strand, the primer’s 3’ OH group allows DNA polymerase to begin DNA synthesis

Action of DNA polymerase

adds nucleotides to existing nucleic acids strand, needs primer (3’ OH)

how to extend the polynucleotide chain

DNA polymerase extends new DNA chains in 5’ → 3’ direction using primer

leading strand

synthesizes 5’ → 3’ towards fork, continuous

lagging strand

synthesizes 5’ → 3’ away from fork, discontinuous, requires multiple RNA primers, produces Okazaki Fragments

how to fill gap in lagging strand

RNA primers removed and resulting gaps filled with DNA (DNA polymerase I), DNA ligase seals nicks

how to deal with incorrectly incorporated nucleotides

polymerase activity allows rapid DNA synthesis, exonuclease activity provides proofreading

type I topoisomerase

cuts one DNA strand, passes other strand through break, and reseals nick

type II topoisomerase

cuts both strands, passes another DNA segment through break, and reseals break

take care of supercoils

type I and II topoisomerases

Ciprofloxacin

inhibits DNA gyrase (topoisomerase II), preventing resealing of DNA breaks

Quinolone

Inhibits topoisomerase II, causing DNA damage and cell death

Enzymes at replication fork

DnaB helicase, SSB, DnaG primase, DNA polymerase III and I, DNA ligase, and DNA topoisomerase

eukaryotic DNA replication versus prokaryotic DNA replication

eukaryotic has multiple origins, S phase, multiple polymerases, and multiple enzymes

eukaryotic DNA organization

packaged with proteins to form chromatin

telomerase

ribonucleoprotein enzyme with reverse transcriptase activity that binds to 3’ end and adds telomeric repeats

telomerase in cancer cells

high telomerase activity causes unlimited replication

nucleoside analogs

modification of OH group at 3’ C of deoxyribose blocking DNA synthesis; found in antiviral and anticancer dugs

reverse transcription

enables retroviruses to convert RNA into DNA

chemotherapeutic agents

target DNA synthesis by inhibiting enzymes or damaging DNA to kill cancer cells (methotrexate, 5-flurouracil, cisplatin)

nucleosome

histone octamers + DNA form ‘beads on a string’ (10nm)