7.

013 Lecture 5: Molecular Biology I 2/12/2016

Section 13.3: How is DNA replicated?
Meselson and Stahl showed that DNA replication is semiconservative: each
parent DNA strand serves as a template for a new strand. A complex of
proteins, most notably DNA polymerase, is involved in replication. New DNA
is polymerized in one direction only, and since the two strands are
antiparallel, one strand is made continuously and the other is synthesized in
short Okazaki fragments that are eventually joined.
• Meselson and Stahl showed that DNA undergoes
semiconservative replication. Each parent strand acts as a
template for the synthesis of a new strand; thus the two replicated
DNA molecules each contain one parent strand and one newly
synthesized strand.
• In DNA replication, the enzyme DNA polymerase catalyzes the
addition of nucleotides to the 3′ end of each strand. Which
nucleotides are added is determined by complementary base
pairing with the template strand.
• The pre-replication complex is a huge protein complex that attaches
to the chromosome at the origin of replication (ori).
• Replication proceeds from the origin of replication on both strands
in the 5′-to-3′ direction, forming a replication fork.
• Primase catalyzes the synthesis of a short RNA primer to which
nucleotides are added by DNA polymerase.
• Many proteins assist in DNA replication. DNA helicase separates
the strands, and single-strand binding proteins keep the strands
from reassociating.
• The leading strand is synthesized continuously and the lagging
strand in pieces called Okazaki fragments. The fragments are
joined together by DNA ligase.
• The speed with which DNA polymerization proceeds is attributed to
the processive nature of DNA polymerases, which can catalyze
many polymerizations at a time. A sliding DNA clamp helps
ensure the stability of this process.
• At the ends of eukaryotic chromosomes are regions of repetitive
DNA sequence called telomeres. Unless the enzyme telomerase
is present, a short segment at the end of each telomere is lost each
time the DNA is replicated. After multiple cell cycles, the telomeres
shorten enough to cause chromosome instability and cell death.
Today
 • Molecular biology: biochemistry of genetic information transfer
• Genetic Information ! hereditary (DNA!RNA!protein) = DNA !
genes
• "replicated (DNA) ! transferred (RNA) ! utilized (proteins)

DNA Replication
• DNA encoded using base sequence = hereditary information/genes
• Base sequences are organized into genes
• Genes are organized into chromosomes bound up with proteins
o Each have thousands of genes
• Nuclear genome replicates once per cell cycle

Rules of Nucleic Acids

1. Complementarity
o A-T (or U), C-G
2. Strands come together in antiparallel fashion such that the 5’-3’ is
parallel to 3’-5’
3. 3’OH addition – nucleotides add to 3’OH
4. Double stranded DNA can denature and renature.

Basic Idea of DNA Replication

• Chromosomes and the DNA on them replicate from defined start
points (origins of replication) abbreviated “ori.”
• Eukaryotic replication takes place at about 100 bases/second.
• A chromosome can have 100 million base pairs (“BP”) of DNA.
• 1 start site for replication – time ~ 300 hours
 o Actual time is 10 hours implying many ori’s
o AT rich – easily denatured (compared to GC)
# Origin recognition complex of proteins bind to ori !
required for replication

Recipe for Replication

• Need the nucleotides, for DNA these are called dNTPs
• Then you need the major enzyme, DNA polymerase (DNA POL)
o This makes the polymer using the template of the particular
DNA strand that is the template
o using an existing strand/primer, adds onto the 3’OH
• DNA polymerase adds to 3’OH
• Enzymes involved:
o helicase: unwinds double stranded DNA
o topoisomerase: prevents ‘knots’ (supercoils) in the
unwinding DNA
o single stranded DNA binding proteins: keep double
stranded DNA apart
o clamp: tethers DNA polymerase on template

In a replication fork - DNA replication from ori

• Template strands elongate to form the new strand differently
• Leading strand: continuous replication from 1 primer
o template is present continuously
• Lagging strand: discontinuous replication
o Many primers, many DNA pieces because template is revealed
sequentially (eventually all the pieces get joined up)