Molecular Basis for

1
Molecular Basis for Relationship between Genotype
and Phenotype
DNA
genotype
DNA sequence
transcription
replication
RNA
translation
amino acid sequence
protein
function
organism
phenotype
2
Overview of DNA Synthesis
DNA polymerases synthesize new strands in 5 to
3 direction. Primase makes RNA primer. Lagging
strand DNA consists of Okazaki fragments. In E.
coli, pol I fills in gaps in the lagging strand
and removes RNA primer. Fragments are joined by
DNA ligase.
3
DNA Replication at Growing Fork
DNA polymerases add nucleotides in 5 to 3
direction. Because of antiparallel
nature, synthesis of DNA is continuous for one
strand and discontinuous for the other strand.
4
DNA Replication Synthesis of Lagging
Strand Several components and steps are involved
in the discontinuous synthesis of the lagging
strand. Note that DNA polymerases move in 3
to 5 direction on the template DNA sequence.
5
DNA Replication Synthesis of Lagging Strand DNA
extended from primers are called Okazaki
fragments. In E. coli, pol I removes RNA
primers and fills in the gaps left in lagging
strands. DNA ligase joins these pieces.
6
Replisome and Accessory Proteins
Looping of template DNA for the lagging strand
allows the two new strands to be synthesized by
one dimer.
pol III holoenzyme is a complex of many different
proteins.
Refer to Figure 7-20 from Introduction to Genetic
Analysis, Griffiths et al., 2012.
7
Priming DNA Synthesis
DNA polymerases can extend (but cannot start) a
chain.
Primase enzyme makes short RNA primer sequence
complementary to template DNA.
DNA polymerase extends RNA primer with DNA.
Primosome is a set of proteins that are involved
in the synthesis of RNA primers.
Refer to Figure 7-20 from Introduction to Genetic
Analysis, Griffiths et al., 2012.
8
Supercoiling results from separation of template
strands during DNA replication.
9
Helicases and Topoisomerases
Helicase enzymes disrupt hydrogen bonding between
complementary bases. Single-stranded binding
protein stabilizes unwound DNA. Unwound
condition increases twisting and coiling, which
can be relaxed by topoisomerases (such as DNA
gyrase).
Topoisomerases can either create or relax
supercoiling. They can also induce or remove
knots.
10
Chromatin assembly factor I (CAF-I) and histones
are delivered to the replication fork. CAF-I and
histones bind to proliferating cell nuclear
antigen (PCNA), the eukaryotic version of clamp
protein.
Nucleosome assembly follows thereafter.
Refer to Figure 7-23 from Introduction to Genetic
Analysis, Griffiths et al., 2012.
11
Overview of DNA Synthesis
DNA polymerases synthesize new strands in 5 to
3 direction. Primase makes RNA primer. Lagging
strand DNA consists of Okazaki fragments. In E.
coli, pol I fills in gaps in the lagging strand
and removes RNA primer. Fragments are joined by
DNA ligase.
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13
Initiation at Origin of Replication
Prokaryotes Fixed origin DnaA proteins DnaB
(helicase) Eukaryotes Multiple origins ORC
protein complex Cdc6 and Cdt1 MCM complex
(helicase)