Chapter 12

Mismatch Repair

Mismatch-repair enzymes recognize the deformity in secondary structure caused by the mismatched base. • Replaces the mismatched base with the correct one

DNA synthesis differs at the ends of linear vs. circular chromosomes (2)

Primers at the ends of chromosomes cannot be replaced, because there is no adjacent 3'-OH to which DNA nucleotides can be attached.

note 4: concept

Replication is initiated at a replication origin, where an initiator protein binds and causes a short stretch of DNA to unwind. DNA helicase breaks hydrogen bonds at replication fork, and single stranded binding proteins stabilize the separated strands. DNA gyrase reduces the torsional strain that develops as the two strands of double helical DNA unwind.

rolling-circle replication

Replication is initiated by a break in one of the nucleotide strands. DNA synthesis begins at the 3' end of the broken strand; the inner strand is used as a template. The 5' end of the broken strand is displaced. It takes place in some viruses and in the F factor of E. coli.

replicon

The amount of DNA replicated from a single origin

bidirectional replication

process where two replication forks proceed outward in both direction simultaneously unwinding and replicating the DNA until they eventually meet.

Meselson and Stahl's Experiment

proved that DNA replication of E. Coli is semiconservative

DNA gyrases (topoisomerases)

reduce supercoiling of the DNA ahead of replication fork by making, then resealing breaks in the DNA backbone.

semiconservative replication

replication in which the two nucleotide strands of DNA separate, each serving as a template for the creation of a complementary strand by bringing in individual nucleotides to base pair with their complementary base on the template

theta replication

replication proceeds around the circle in both directions from the origin; - replication is bidirectional. Found in E.coli

conservative replication

the entire double-stranded DNA molecule serves as a template for a while new molecule of DNA, and the original DNA molecule is fully conserved during replication

The Holliday model

the point at which nucleotide strands pass from one DNA molecule to the other

DNA polymerase III

(at least 10 different polypeptide subunits): elongates primers in the 5' to 3' direction, giving continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand.

Linear Eukaryotic Replication

- Each chromosome contains numerous origins. • At each origin, the DNA unwinds producing a replication bubble. • DNA synthesis takes place on both strands at each end of the bubble as replication forks proceed outward. • Eventually, the forks of adjacent bubbles run into each other and the segments of DNA fuse, producing two identical linear DNA molecules.

DNA Replication in Eukaryotes

- Eukaryotic chromosomes contain multiple replication origins b/c - Eukaryotes have more DNA to replicate than prokaryotes. - Eukaryotic DNA polymerase is slower than that of prokaryotes.

Telomerase

- a ribonucleoprotein (RNA and protein) reverse transcriptase • Its RNA has sequence complementary to telomere sequence. • Uses its own RNA as template. • Adds additional telomeric repeats to the ends of telomeres. page 341 - 12.19 pic

Each active replication fork requires five basic components:

1) Helicase to unwind the DNA. 2) Single-stranded-binding proteins to keep the nucleotide strands separate long enough to allow replication. 3) The topoisomerase gyrase to remove strain ahead of the replication fork. 4) Primase to synthesize primers with a 3'-OH group at the beginning of each DNA fragment. 5) DNA polymerase to synthesize the leading and lagging nucleotide strands.

DNA purpose 1

1) it serves as a template for the synthesis of the new DNA - the template determines the sequence of the new DNA strand, through the specificity of base pairing

3 major Requirements of Replication

1. A template consisting of single-stranded DNA. 2. Substrates (raw materials) to be assembled into a new nucleotide strand. 3. Enzymes and other proteins that "read" the template and assemble the substrates into a DNA molecule

3 differences beween prokaryotic and eukaryotic replication:

1. greater size of eukaryotic genomes which requires replication to be initiated at multiple origins. 2. eukaryotic chromosomes are linear whereas prokaryotic chromosomes are circular 3. DNA template is associated with histone proteins in the form of nucleosomes, and nucleosomes assembly must immediately fallow DNA replication.

DNA purpose 2

2) it serves as a primer for DNA synthesis. - It turns out that DNA polymerase I cannot initiate DNA synthesis without having a free 3'-OH to add a new nucleotide to. - DNA synthesis therefore needs a primer, a preexisting piece of nucleic acid to serve as an initiator of DNA synthesis.

DNA synthesis count..

A phosphodiester bond forms between the two nucleotides and phosphate ions are released. - H-bonds form between complementary base pairs.

Direction of Replication

Because the two single-stranded DNA templates are anti-parallel and strand elongation is always 5'-to-3', replication on the two strands proceeds in opposite directions.

Initiator proteins

Bind to origin and separates strands of DNA to initiate replication.

Note:

Circular DNA molecules that undergo theta or rolling-circle replication have a single origin of replication. However, because eukaryotic cells contain much more DNA than bacteria, there must be multiple origins of replication on each chromosome in order to replicate all of the DNA in a timely fashion.

rolling-circle replication count...

Cleavage releases a single-stranded linear DNA and a double stranded circular DNA.The linear DNA may circularize and serve as a template for synthesis of a complementary strand.

note 5:

DNA polymerase III and I carry out DNA synthesis in replication; the other 3 have specialized functions in DNA repair.

Nucleotide Selection

DNA polymerases are very particular in pairing nucleotides with their complements on the template strand.

note 2:

DNA synthesis is always 5' to 3'

note 3:

DNA synthesis takes place simultaneously, but in opposite directions on the two DNA template strands. - However, DNA synthesis is continuous on one template strand of DNA and discontinuous on the other.

theta replication model

DNA template: circular breaks of nucleotide strands: no # of replicons: 1 both: unidirectional and bidirectional product: 2 circular molecules

Rolling circle replication model

DNA template: circular breaks of nucleotide strands: yes # of replicons: 1 both: unidirectional product: 1 circular molecule and one linear molecule that may circularize

Linear Eukaryotic replication model

DNA template: linear breaks of nucleotide strands: no # of replicons: many both: bidirectional product: 2 linear molecules

Helicases

Denature DNA at replication fork. Comes in after initiator protein and binds to the lagging strand template

end-replication problem

Every time a linear chromosome replicates, the lagging strand at each end gets shorter because there is a minimum length of DNA needed for initiation of an Okazaki fragment. - next 3 slides explains it in details

Does recombination due to crossing over occur before or after DNA synthesis?

Genetic evidence suggests that crossing over takes place after DNA synthesis.

what are Hybrid chromatids called?

Harlequin chromosomes

Proofreading

If a noncomplementary (incorrect) nucleotide is added to a chain by mistake, DNA polymerase will detect the error and use its 3'-to- 5' exonuclease activity to remove the incorrect nucleotide. - If an incorrect base is added the DNA polymerase is stalled and it removes the incorrect base, replacing it with the correct one. Then replication proceeds.

DNA synthesis differs at the ends of linear vs. circular chromosomes (1)

In linear DNA with multiple origins of replication, elongation of DNA in adjacent replicons provides a 3'-OH group for replacement of each primer.

Rolling-circle Replication conclusion

The products of rolling- circle replication are multiple circular DNA molecules.

fidelity replication conclusion:

The sequential application of multiple mechanisms ensures highly accurate DNA replication.

DNA synthesis differs at the ends of linear vs. circular chromosomes (3)

When the primer at the end of a chromosome is removed, there is no 3'-OH group to which DNA nucleotides can be attached, producing a gap.

Primase

an RNA polymerase that uses DNA as a template to make RNA primer for DNA replication. Primase synthesizes short stretches of RNA nucleotides, providing a 3'-OH group to which DNA polymerase can add DNA nucleotides.

Single-strand binding proteins

attaches tightly to exposed single stranded DNA after unwound by helicase. it keep DNA single stranded and prevents hairpins formations.

origin of replication

bacterial replication begins at the same spot on the chromosome every time

dispersive replication

both nucleotides break down into fragments, which serves as a template for new synthesis of new DNA fragments. Thus suggested that each DNA molecule after replication might consist of segments of new and old DNA interspersed.

DNA polymerase I:

digests RNA primers with its 5' 3' exonuclease activity and replaces with DNA - fallows polymerase III

DNA polymerases

family of enzymes that catalyzes DNA replication. can add nucleotides only to the 3' end of the growing strand (not the 5' end)

DNA polymerase II:

involves in DNA repair

DNA ligase

joins unlinked DNA strands by sealing nicks in the sugar-phosphate backbone

note 5:

looks over/ memorize table 12.4 on page 334 - the previous information was regarding bacterial replication

DNA synthesis

new DNA is synthesized from deoxynucleoside triphosphates (dNTPs). - In replication, the 3'-OH group of the last nucleotide on the strand attacks the 5'-phosphate group of the incoming dNTP. - Two phosphates are cleaved off.

Despite diferences all of E.coli's DNA polymerases: (count..)

• Catalyze the formation of a phosphodiester bond by joining the 5'-phosphate group of the incoming nucleotide to the 3'-OH group of the proceeding nucleotide on the growing strand, cleaving off two phosphates in the process. • Produce newly synthesized strands that are complementary and anti-parallel to the template strands. • Are associated with a number of other proteins (enzymes).

Replication of Chromosome Ends

• More nucleotides are added. • The telomerase is removed. • Synthesis takes place on the complementary strand, filling in the gap due to removal of the RNA primer at the end.

Eukaryotic DNA Polymerases:

• Multiple DNA polymerases (at least 13) have been identified in eukaryotes: • α, δ, ε - are essential for DNA replication • β and θ - DNA repair • γ - mtDNA synthesis

3 processes that prevent DNA replication errors

• Nucleotide selection. • DNA proofreading. • Mismatch repair.

telomerase aging and disease

• Shortening of telomeres appears to contribute to cell death and aging. • Germ-line cells: high telomerase activity • Somatic cells: very low telomerase activity • Telomerase negative mice: premature aging after a # of generations. • Somatic cells engineered to have telomerase activity: divide indefinitely. • Telomerase activation is part of the development of most advanced cancers.

Despite diferences all of E.coli's DNA polymerases:

• Synthesize any sequence specified by the template strand. • Synthesize in the 5'to3' direction by adding nucleotides to a 3'-OH group. • Use dNTPs to synthesize new DNA. • Require a primer (w/3'-OH) to initiate synthesis.

Replication of Chromosome Ends 2

• The G-rich single- strand that has been extended by telomerase may fold back on itself. • This forms a terminal loop by nonconventional base pairing. • This loop provides a 3'-OH group for attachment of DNA nucleotides.