Chapter 14 Extrachromosomal Replication Jocelyn E Krebs Figure

Chapter 14 Extrachromosomal Replication Jocelyn E. Krebs

Figure 14. CO: A model depicting pilus assembly in bacteria. Photo courtesy of Dr. Han Remaut* and Gabriel Waksman, ISMB - UK. [* present address: Structural & Molecular Microbiology, VUB / VIB, Belgium]

14. 1 Introduction • Extrachromosomal DNA-plasmid, Phage • 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. 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. • •

Figure 14. 01: Several types of independent genetic units exist in bacteria.

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?

Problem: in lagging strand, DNA pol 5 3 activity, primer Solution; converting liner to circular or multimeric DNA creating hairpin eliminates free end multi-copies of telomere special protein (terminal protein) 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 ds. DNA 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. • This protein binds 9 -18 bp from end (17 -48 region is essential for host protein binding , nuclear factor I

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 cis-acting relaxase that generates singlestranded circles from the tail produced by rolling circle replication. Figure 14. 09: yx 174 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. 100 kb • Integration into host genome by recombination in IS sequence • ori. V replication origin of plasmid-form F-plasmid

• transfer region – A segment on the F plasmid that is required for bacterial conjugation (33 kb, 40 genes). • Tra D, I, M, Y; directly involved in DNA transfer • Others ; type 4 secretion system • 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.

• • The F plasmid encodes a DNA translocation complex and specific pili that form on the surface of the bacterium. Pilus : hair-like structure (2 -3 u. M long, hollwo cylinder, 8 nm diameter, 2 nm hole) pilin – The subunit that is polymerized into the pilus in bacteria (encoding by tra. A). 12 genes are involved in pili formation An F-pilus enables an F-positive bacterium to contact an F-negative bacterium and to initiate conjugation. Tra. S and Tra. T: surface exclusion F-pili can increase suspecitibility of infection by DNA or RNA phage (F- bacteria is resistant) Initial contact is easily broken. But repeated trial can make close contact Tra D; coupling protein (necessary for recruitment of plasmid DNA to the T 4 SS) Figure 14. 11: Mating bacteria are initially connected when donor F pili contact the recipient bacterium. © Dennis Kunkel/Phototake, Inc. /Alamy Images

14. 7 Conjugation Transfers Single-Stranded DNA • • • Transfer of an F plasmid is initiated when rolling circle replication begins at ori. T. Tra. M recognizes mating pair Tra. Y binds ori. T and recruits Tra. I (relexase; similar to A protein and helicase) form the relaxosome (with integration-host factor; IHF) The formation of a relaxosome initiates transfer into the recipient bacterium. The transferred DNA is converted into doublestranded form in the recipient bacterium.

• 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 ori. T. 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. • Multi-copy plasmids exist at >1 plasmid copy per bacterial chromosome origin random segregation.

Single copy partition system par. A; ATPase par. B and par. A complex binds to par. S site (DNA region; centromere-like) Figure 14. 14: A common segregation system consists of genes par. A and par. B and the target site par. S.

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. • Par. R=Par. B • Par. M=ATPase (actin-like) Figure 14. 15: The partition of plasmid R 1 involves polymerization of the Par. M 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.

• 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 Vir. A is autophosphorylated on histidine when it binds an inducer and activates Vir. G 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.

• 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.

• The T-DNA is transferred as a complex of singlestranded DNA with the Vir. E 2 single strand-binding protein. • The single-stranded T-DNA is converted into doublestranded 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? • mt. DNA 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.