STEPTRACKERBIOCHEMISTRY + PHARMACOLOGY + MICROBIOLOGY | MOLECULAR BIOLOGY | HIGH-YIELD REVIEW

DNA Replication

Semiconservative copying at the replication fork — each enzyme has a job, and many are antibiotic or chemotherapy targets.

FOCUS ON

Synthesis is always 5′ → 3′; the template is read 3′ → 5′. Leading strand is continuous; lagging strand is made as Okazaki fragments, each needing an RNA primer. Proofreading is 3′ → 5′ exonuclease. Prokaryotes: DNA pol III builds, pol I removes primers. Topoisomerases relieve supercoils (fluoroquinolones, etoposide, irinotecan). Telomerase adds TTAGGG to chromosome ends.

ENZYME · PROTEIN

JOB

PROKARYOTE vs EUKARYOTE

DRUG · CLUE

Origin of replication

AT-rich start sites where strands separate

Single origin (circular) vs many origins (linear chromosomes)

A–T pairs have 2 H-bonds → easier to open

Helicase

Unwinds the double helix at the fork (uses ATP)

Both

Defective helicases: Bloom, Werner syndromes

Single-strand binding proteins

Keep separated strands apart

Both

—

Topoisomerase I

Single-strand nick to relieve supercoils, then reseals

Both

Human: irinotecan, topotecan

Topoisomerase II

gyrase in bacteria

Double-strand break to relieve (or add negative) supercoils

Bacterial gyrase (II) and topo IV

Bacteria: fluoroquinolones · human: etoposide, teniposide, anthracyclines

Primase

Lays down short RNA primers for DNA polymerase to extend

Both

DNA polymerase can't start a strand de novo

DNA polymerase III

prokaryotes only

Elongates leading and lagging strands 5′→3′; 3′→5′ proofreading

Eukaryotic equivalents: pol δ (lagging/leading), pol ε

Main replicative enzyme in bacteria

DNA polymerase I

prokaryotes only

Removes RNA primer (5′→3′ exonuclease) and fills the gap; also proofreads

Eukaryotes use RNase H + FEN1

Only pol I has 5′→3′ exonuclease activity

DNA ligase

Seals nicks between Okazaki fragments (phosphodiester bond)

Both

Also used in repair

Telomerase

Reverse transcriptase with its own RNA template; adds TTAGGG repeats at 3′ ends

Eukaryotes only (linear chromosomes)

Active in stem cells, germ cells, most cancers; shortening = aging

Reverse transcriptase

RNA → DNA (retroviruses, hepatitis B)

Viral

NRTIs, NNRTIs

Chain terminators

Nucleoside analogs lacking a 3′-OH stop elongation

Viral and human polymerases

Acyclovir, zidovudine, cytarabine

Picture it — the replication fork

Replication forkHelicase opens the helix and topoisomerase relieves the twist ahead of it. The leading strand is built continuously toward the fork; the lagging strand is built in Okazaki fragments, each starting from an RNA primer, and joined by ligase.

Schematic diagrams, not to scale.

Mechanism — at the replication fork

HelicaseOpens

→

SSB + topoisomeraseHold open, relieve twist

→

PrimaseRNA primer

→

DNA pol III5′→3′ synthesis

→

Pol I + ligaseReplace primer, seal

The lagging strand runs away from the fork, so it is made backward in pieces (Okazaki fragments) that ligase joins.

Topoisomerase drugs

DRUG

TARGET

CLASSIC TOXICITY

Ciprofloxacin, levofloxacin

Bacterial gyrase + topo IV

Tendon rupture, QT prolongation

Irinotecan, topotecan

Topo I

Diarrhea, myelosuppression

Etoposide, teniposide

Topo II

Myelosuppression, alopecia

Doxorubicin

Topo II + free radicals

Dilated cardiomyopathy

How it's tested

COMMON CONFUSIONDNA pol I removes primers using a 5′→3′ exonuclease; proofreading by any polymerase is 3′→5′ exonuclease.

THE TRAP"Which enzyme activity is unique to prokaryotic pol I?" — 5′→3′ exonuclease (primer removal), not proofreading.

YOUR APPROACHOrder the fork events (open → stabilize → untwist → prime → extend → replace primer → seal) and map each drug to its step.

If you see… think…

Antibiotic inhibiting DNA gyrase → fluoroquinolone

Chemo agent inhibiting topo I, severe diarrhea → irinotecan

Chemo agent inhibiting topo II → etoposide

Enzyme that lays RNA primers → primase

Removes RNA primer in bacteria → DNA polymerase I

Joins Okazaki fragments → DNA ligase

Enzyme keeping cancer cells immortal → telomerase

Repeat added to chromosome ends → TTAGGG

Proofreading activity → 3′→5′ exonuclease

Antiviral lacking a 3′-OH → chain terminator