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 single-stranded 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 Figure 14.09: yx174 RF DNA is a template for synthesizing single-stranded viral circles. 14.5 Rolling Circles Are Used to Replicate Phage Genomes The φX A protein is a cis-acting relaxase that generates single-stranded circles from the tail produced by rolling circle replication.

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 Single-Stranded 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, Agrobacterium-mediated genetic transformation of plants, Curr. Opin. Biotechnol . 17, pp. 147–154. Copyright 2006, and with permission from Elsevier (http://www.sciencedirect.com/science/journal/09581669).

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.