Репликация

Сентябрь 3, 2022

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4. Nick translation describes the ability of E. coli DNA polymerase I to use a nick as
5. The common organization of DNA polymerases has a palm that contains the catalytic site, fingers that
6. The crystal structure of phage T7 DNA polymerase shows that the template strand takes a sharp
7. The leading strand of DNA is synthesized continuously in the 5’-3’ direction. The lagging strand of
8. A helicase is an enzyme that uses energy provided by ATP hydrolysis to separate the strands
9. A hexameric helicase moves along one strand of DNA. It probably changes conformation when it binds
10. Priming is required to start DNA synthesis Key Terms A primer is a short sequence (often
11. Coordinating synthesis of the lagging and leading strands Key Concepts Different enzyme units are required to
12. DNA polymerase III holoenzyme assembles in stages, generating an enzyme complex that synthesizes the DNA of
13. The clamp controls association of core enzyme with DNA · The core on the leading strand
17. Okazaki fragments are linked by ligase DNA ligase makes a bond between an adjacent 3’-OH and
18. Similar functions are required at all replication forks
19. Репликационный глазок The bacterial genome is a single circular replicon
20. Creating the replication forks at an origin The origin of E. coli,oriC, is 245 bp in
21. Терминация – встреча вилок
23. Скачать презентацию

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Nick translation describes the ability of E. coli DNA polymerase I

to use a nick as a starting point from which one strand of a duplex DNA can be degraded and replaced by resynthesis of new material; is used to introduce radioactively labeled nucleotides into DNA in vitro.
DNA polymerase I has a unique 5’–3’ exonuclease activity that can be combined with DNA synthesis to perform nick translation.

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The common organization of DNA polymerases has a palm that contains

the catalytic site, fingers that position the template, a thumb that binds DNA and is important in processivity, an exonuclease domain with its own active site, and an N-terminal domain.

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The crystal structure of phage T7 DNA polymerase shows that the

template strand takes a sharp turn in order to be exposed to the incoming nucleotide.

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The leading strand of DNA is synthesized continuously in the 5’-3’

direction.
The lagging strand of DNA must grow overall in the 5’-3’ direction and is synthesized discontinuously in the form of short fragments (5’-3’ ) that are later connected covalently.
Okazaki fragments are the short stretches of 1000-2000 bases produced during discontinuous replication; they are later joined into a covalently intact strand.
Semidiscontinuous replication is mode in which one new strand is synthesized continuously while the other is synthesized discontinuously.

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A helicase is an enzyme that uses energy provided by ATP

hydrolysis to separate the strands of a nucleic acid duplex.
The single-strand binding protein (SSB) binds to single-stranded DNA, thereby preventing the DNA from forming a duplex.

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A hexameric helicase moves along one strand of DNA. It probably

changes conformation when it binds to the duplex, uses ATP hydrolysis to separate the strands, and then returns to the conformation it has when bound only to a single strand.

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Priming is required to start DNA synthesis
Key Terms
A primer is a

short sequence (often of RNA) that is paired with one strand of DNA and provides a free 3’-OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.
The primase is a type of RNA polymerase that synthesizes short segments of RNA that will be used as primers for DNA replication.
Key Concepts
All DNA polymerases require a 3’ –OH priming end to initiate DNA synthesis.
The priming end can be provided by an RNA primer, a nick in DNA, or a priming protein.
For DNA replication, a special RNA polymerase called a primase synthesizes an RNA chain that provides the priming end.
Priming of replication on double-stranded DNA always requires a replicase, SSB, and primase.
DnaB is the helicase that unwinds DNA for replication in E. coli.

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Coordinating synthesis of the lagging and leading strands
Key Concepts
Different enzyme units

are required to synthesize the leading and lagging strands.
In E. coli both these units contain the same catalytic subunit (DnaE).
In other organisms, different catalytic subunits may be required for each strand.
DNA polymerase holoenzyme has 3 subcomplexes
The clamp loader is a 5 subunit protein complex which is responsible for loading the ß clamp on to DNA at the replication fork.
The E. coli replicase DNA polymerase III is a 900 kD complex with a dimeric structure.
Each monomeric unit has a catalytic core, a dimerization subunit, and a processivity component.
A clamp loader places the processivity subunits on DNA, and they form a circular clamp around the nucleic acid.
One catalytic core is associated with each template strand.

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DNA polymerase III holoenzyme assembles in stages, generating an enzyme complex

that synthesizes the DNA of both new strands.

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The clamp controls association of core enzyme with DNA
· The core

on the leading strand is processive because its clamp keeps it on the DNA.
· The clamp associated with the core on the lagging strand dissociates at the end of each Okazaki fragment and reassembles for the next fragment.
· The helicase DnaB is responsible for interacting with the primase DnaG to initiate each Okazaki fragment.

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Okazaki fragments are linked by ligase
DNA ligase makes a bond between

an adjacent 3’-OH and 5’-phosphate end where there is a nick in one strand of duplex DNA.
Each Okazaki fragment starts with a primer and stops before the next fragment.
DNA polymerase I removes the primer and replaces it with DNA in an action that resembles nick translation.
DNA ligase makes the bond that connects the 3’ end of one Okazaki fragment to the 5’ beginning of the next fragment.

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Similar functions are required at all replication forks

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Репликационный глазок
The bacterial genome is a single circular replicon

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Creating the replication forks at an origin
The origin of E. coli,oriC,

is 245 bp in length
Origins in S. cerevisiae are short A·T-rich sequences that have an essential 11 bp sequence.
The ORC is a complex of 6 proteins that binds to an ARS.
Initiation at oriC requires the sequential assembly of a large protein complex.
DnaA binds to short repeated sequences and forms an oligomeric complex that melts DNA.
6 DnaC monomers bind each hexamer of DnaB and this complex binds to the origin.
A hexamer of DnaB forms the replication fork.
A short region of A·T-rich DNA is melted.
Gyrase and SSB are also required.

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