Transcript Chapter 14

Chapter 14
Extrachromosomal Replication
14.1 Introduction
• plasmid – Circular, extrachromosomal DNA.
– It is autonomous and can replicate itself.
• temperate phage – A phage that can enter a
lysogenic cycle within the host (can become a
prophage integrated into the host genome).
• lysogenic – The ability of a phage to survive in
a bacterium as a stable prophage component of
the bacterial genome.
14.1 Introduction
• episome – A plasmid able to integrate into bacterial
DNA.
• immunity – In plasmids, the ability of a plasmid to
prevent another of the same type from becoming
established in a cell.
14.2 The Ends of Linear DNA Are a
Problem for Replication
• Special arrangements must be made to replicate the
DNA strand with a 5′ end.
Figure 14.02: Replication could run off the 3‘ end of a newly synthesized linear strand, but
could it initiate at a 5‘ end?
14.3 Terminal Proteins
Enable Initiation at the
Ends of Viral DNAs
• strand displacement – A
mode of replication of some
viruses in which a new DNA
strand grows by displacing
the previous (homologous)
strand of the duplex.
Figure 14.03: Adenovirus DNA
replication is initiated separately at
the two ends of the molecule and
proceeds by strand displacement.
14.3 Terminal Proteins Enable Initiation at
the Ends of Viral DNAs
• A terminal protein binds to the 5′ end of DNA and
provides a cytidine nucleotide with a 3′–OH end that
primes replication.
• The dsDNA viruses adenovirus and φ29 have terminal
proteins that initiate replication by generating a new 5′
end.
• The newly synthesized strand displaces the
corresponding strand of the original duplex.
• The released strand base pairs at the ends to form a
duplex origin that initiates synthesis of the
complementary strand.
14.3 Terminal Proteins Enable Initiation at
the Ends of Viral DNAs
Figure 14.05: Adenovirus terminal protein binds to the 5' end of DNA and provides a C-OH
end to prime synthesis of a new DNA strand.
14.4 Rolling Circles Produce Multimers of a
Replicon
• A rolling circle generates single-stranded multimers of
the original sequence.
Figure 14.06: The rolling
circle generates a
multimeric singlestranded tail.
Figure 14.08: The
fate of the
displaced tail
determines the
types of products
generated by
rolling circles.
14.5 Rolling Circles Are
Used to Replicate
Phage Genomes
• The φX A protein is a cisacting relaxase that
generates single-stranded
circles from the tail produced
by rolling circle replication.
Figure 14.09: yx174 RF DNA is a
template for synthesizing singlestranded viral circles.
14.6 The F Plasmid Is Transferred by
Conjugation between Bacteria
• conjugation – A process in which two cells
come in contact and transfer genetic material.
– In bacteria, DNA is transferred from a donor to a
recipient cell.
• A free F plasmid is a replicon that is maintained
at the level of one plasmid per bacterial
chromosome.
14.6 The F Plasmid Is Transferred by
Conjugation between Bacteria
• transfer region – A segment on the F plasmid that is
required for bacterial conjugation.
• An F plasmid can integrate into the bacterial
chromosome, in which case its own replication system is
suppressed.
Figure 14.10: The tra region of the F plasmid contains the genes needed
for bacterial conjugation.
14.6 The F Plasmid Is Transferred by
Conjugation between Bacteria
• The F plasmid encodes a DNA translocation complex
and specific pili that form on the surface of the
bacterium.
• pilin – The subunit that is polymerized into the pilus in
bacteria.
• An F-pilus enables an F-positive bacterium to contact an
F-negative bacterium and to initiate conjugation.
14.7 Conjugation Transfers Single-Stranded
DNA
• Transfer of an F plasmid is initiated when rolling circle
replication begins at oriT.
• The formation of a relaxosome initiates transfer into the
recipient bacterium.
• The transferred DNA is converted into double-stranded
form in the recipient bacterium.
14.7 Conjugation
Transfers SingleStranded DNA
• When an F plasmid is free,
conjugation “infects” the
recipient bacterium with a
copy of the F plasmid.
Figure 14.13: Transfer of
chromosomal DNA occurs when an
integrated F factor is nicked at oriT.
14.7 Conjugation Transfers Single-Stranded
DNA
• When an F plasmid is integrated, conjugation
causes transfer of the bacterial chromosome
until the process is interrupted by (random)
breakage of the contact between donor and
recipient bacteria.
• Hfr – A bacterium that has an integrated F
plasmid within its chromosome.
– Hfr stands for high frequency recombination, referring
to the fact that chromosomal genes are transferred
from an Hfr cell to an F– cell much more frequently
than from an F+ cell.
14.8 Single-Copy Plasmids Have a
Partitioning System
• copy number – The number of copies of a plasmid that
is maintained in a bacterium relative to the number of
copies of the origin of the bacterial chromosome.
• Single-copy plasmids exist at one plasmid copy per
bacterial chromosome origin.
• Multicopy plasmids exist at >1 plasmid copy per bacterial
chromosome origin.
14.8 Single-Copy
Plasmids Have a
Partitioning System
• Partition systems ensure
that duplicated plasmids
are segregated to different
daughter cells produced
by a division.
Figure 14.15: The partition of plasmid
R1 involves polymerization of the ParM
ATPase between plasmids.
14.9 Plasmid Incompatibility Is Determined
by the Replicon
• Plasmids in a single compatibility group have origins
that are regulated by a common control system.
Figure 14.16: Two plasmids are incompatible (they belong to the same compatibility
group) if their origins cannot be distinguished at the stage of initiation.
14.10 The Bacterial Ti Plasmid Transfers
Genes into Plant Cells
• In crown gall disease, infection with the bacterium A.
tumefaciens can transform plant cells into tumors.
• The infectious agent is the Ti plasmid carried by the
bacterium.
• The plasmid also carries genes for synthesizing and
metabolizing opines (arginine derivatives) that are used
by the bacterium.
14.10 The Bacterial Ti
Plasmid Transfers Genes
into Plant Cells
• T-DNA, part of the DNA of the
Ti plasmid, is transferred to the
plant cell nucleus, but the vir
genes outside this region are
required for the transfer
process.
Figure 14.18: T-DNA is transferred from
Agrobacterium carrying a Ti plasmid
into a plant cell, where it becomes
integrated into the nuclear genome.
14.10 The Bacterial Ti Plasmid Transfers
Genes into Plant Cells
Figure 14.19: A model for Agrobacterium -mediated genetic transformation.
Reprinted from T. Tzfira and V. Citovsky, Agrobacteriummediated genetic transformation of plants, Curr. Opin.
Biotechnol . 17, pp. 147–154. Copyright 2006, and with
permission from Elsevier
(http://www.sciencedirect.com/science/journal/095816
14.11 Transfer of T-DNA Resembles
Bacterial Conjugation
• The vir genes are induced by phenolic compounds
released by plants in response to wounding.
• The membrane protein VirA is autophosphorylated on
histidine when it binds an inducer and activates VirG by
transferring the phosphate to it.
Figure 14.21: T-DNA has almost identical repeats of 25 bp at each end in the Ti plasmid.
14.11 Transfer of T-DNA Resembles
Bacterial Conjugation
• T-DNA is generated when a nick at the right
boundary creates a primer for synthesis of a new
DNA strand.
• The pre-existing single strand that is displaced
by the new synthesis is transferred to the plant
cell nucleus.
• Transfer is terminated when DNA synthesis
reaches a nick at the left boundary.
14.11 Transfer of T-DNA Resembles
Bacterial Conjugation
Figure 14.22: T-DNA is generated by displacement when DNA synthesis starts at a nick
made at the right repeat. The reaction is terminated by a nick at the left repeat.
14.11 Transfer of T-DNA Resembles
Bacterial Conjugation
• The T-DNA is transferred as a complex of
single-stranded DNA with the VirE2 single
strand-binding protein.
• The single-stranded T-DNA is converted into
double-stranded DNA and integrated into the
plant genome.
• The mechanism of integration is not known.
– T-DNA can be used to transfer genes into a plant
nucleus.
14.12 How Do
Mitochondria Replicate
and Segregate?
• mtDNA replication and
segregation to daughter
mitochondria is stochastic.
• heteroplasmy – Having
more than one mitochondrial
allelic variant in a cell.
• Mitochondrial segregation to
daughter cells is also
stochastic.
Figure 14.23: Mitochondrial DNA replicates by
increasing the number of genomes in proportion
to mitochondrial mass, w/o ensuring that each
genome replicates the same number of times.