DNA replication steps: enzymes, leading and lagging

Key ideas

Base pairing. Adenine (A) pairs with thymine (T), and guanine (G) pairs with cytosine (C). So if you know one strand, you know the other.
Antiparallel strands. The two strands run in opposite directions. One runs 5' to 3', and its partner runs 3' to 5'. The numbers name the ends of the sugar-phosphate backbone.
Semiconservative. Each new double helix has one original strand and one new strand. Meselson and Stahl showed this in 1958.
5' to 3' only. DNA polymerase can only add nucleotides to the 3' end of a growing strand, and it needs a primer to start.

The main enzymes and proteins (OpenStax Biology 2e, 14.4 and 14.5)
Enzyme or protein	Job
Helicase	Unwinds the helix by breaking the hydrogen bonds between base pairs, making a replication fork
Single-strand binding proteins	Coat the separated strands so they do not snap back together
Topoisomerase	Relieves the over-winding (supercoiling) ahead of the fork
Primase	Makes a short RNA primer that gives DNA polymerase a starting point
DNA polymerase	Adds DNA nucleotides to the primer, 5' to 3', and proofreads
Primer removal (DNA pol I in bacteria, RNase H in eukaryotes)	Removes the RNA primers so they can be replaced with DNA
DNA ligase	Seals the gaps between Okazaki fragments

Leading and lagging strands

The fork opens in one direction, but the two templates point opposite ways. On the leading strand, polymerase follows the fork and builds one continuous strand. On the lagging strand, it has to work away from the fork, so it builds short pieces called Okazaki fragments, each starting from its own primer. Ligase then joins them.

Worked examples

Example 1: write the complementary strand

Problem One strand of DNA reads 5'-AGCTTACG-3'. Write the complementary strand, labeled 5' to 3'.

Pair each base: A with T, G with C, C with G, T with A. Reading along, the partner is 3'-TCGAATGC-5'.
Strands are antiparallel, so the partner's 5' end sits opposite the first strand's 3' end. To write it 5' to 3', read the partner backward.

Answer 5'-CGTAAGCT-3'

Example 2: Chargaff's rule

Problem In a sample of double-stranded DNA, 30% of the bases are adenine. What percent are thymine, guanine and cytosine?

Every A pairs with a T, so T = A = 30%.
That leaves $100\% - 30\% - 30\% = 40\%$ for G and C.
G pairs with C, so they split it evenly: 20% each.

Answer T 30%, G 20%, C 20%

Example 3: tracking old strands (Meselson and Stahl)

Problem A DNA molecule with both strands labeled heavy (15N) is copied twice in a medium with only light (14N) nucleotides. How many of the resulting molecules are hybrid (one heavy, one light strand), and how many are fully light?

Round 1: each heavy strand gets a new light partner. 2 molecules, both hybrid.
Round 2: the 4 strands (2 heavy, 2 light) each get a new light partner. The 2 heavy strands make 2 hybrid molecules. The 2 light strands make 2 fully light molecules.

Answer 2 hybrid and 2 fully light molecules out of 4

Common mistakes and how to fix them

Pairing A with U. Uracil is only in RNA. In DNA, A pairs with T.
Forgetting antiparallel direction. Fix: always label the 5' and 3' ends, and remember that the complement's 5' end lines up with the template's 3' end.
Saying the lagging strand is made 3' to 5'. Both strands are made 5' to 3'. The lagging strand is made in pieces because of that rule.
Mixing up helicase and ligase. Helicase opens the helix at the start. Ligase seals gaps at the end.

Practice questions

What is the complementary strand of 5'-GATTACA-3', written 5' to 3'?
1. 5'-CTAATGT-3'
2. 5'-TGTAATC-3'
3. 5'-GATTACA-3'
4. 5'-CUAAUGU-3'
Show answer
Answer: 5'-TGTAATC-3'
Pairing gives 3'-CTAATGT-5'. Read it backward to write it 5' to 3': TGTAATC. The U choice is RNA, not DNA.
Which enzyme unwinds the DNA double helix?
1. Ligase
2. Primase
3. Helicase
4. DNA polymerase
Show answer
Answer: Helicase
Helicase breaks the hydrogen bonds between the bases to open the replication fork.
A double-stranded DNA sample is 18% guanine. What percent is adenine?
Show answer
Answer: 32%
C = G = 18%, so G + C = 36%. A + T = 64%, and A = T, so A = 32%.
A fully heavy (15N) DNA molecule replicates 3 times in light (14N) medium. How many of the 8 molecules still contain a heavy strand?
Show answer
Answer: 2 molecules
The two original heavy strands are never broken up or copied into new heavy DNA. Each sits in its own molecule, so 2 of 8 contain a heavy strand, no matter how many rounds.
On which strand are Okazaki fragments made?
1. Leading strand
2. Lagging strand
3. Both strands equally
4. The RNA primer
Show answer
Answer: Lagging strand
The lagging strand is built away from the fork, so polymerase works in short pieces that ligase later joins.

Frequently asked questions

Why is replication called semiconservative?

Semi means half. Each new DNA molecule conserves, or keeps, half of the original: one old strand plus one new strand. Meselson and Stahl showed this with heavy and light nitrogen. After one round, all DNA was a hybrid band, which ruled out the idea that the original molecule stays whole.

How fast is DNA replication?

OpenStax gives about 1000 nucleotides per second in bacteria and 50 to 100 per second in eukaryotes. Human cells make up for the slower speed by starting at many origins at once, up to about 100,000 across the genome, so S phase can finish in hours.

What happens if DNA polymerase makes a mistake?

DNA polymerase proofreads as it goes and fixes most wrong bases right away. Mismatch repair enzymes catch many of the rest after replication. The few errors that remain become mutations, which can be harmless, harmful or occasionally helpful.