Extrachromosomal replication of DNA

plasmid,mitochondria and chloroplast replications

1. EXTRACHROMOSOMAL REPLICATION OF DNA LUBNA S SUBAIR M.Sc.Biotechnology CUSAT

2. contents Extrachromosomal DNA Prokaryotic extrachromosomal DNA Plasmid Replication mechanisms Eukaryotic extrachomosomal DNA Mitochondria Replication mechanism Chloroplast Replication mechanism

3. Extrachromosomal DNA Extrachromosomal DNA   is any DNA that is found off the chromosomes, either inside or outside the nucleus of a cell.  Most DNA in an individual genome is found in chromosomes contained in the nucleus. The extrachromosomal DNA exist and serve important biological functions . In prokaryotes, nonviral extrachromosomal DNA are primarily found in  plasmids  whereas in eukaryotes extrachromosomal DNA are primarily found in  organelles . In prokaryotes,it occurs outside the nucleoid region as circular or linear plasmids.  Bacterial plasmids are typically short sequences, consisting of 1 kilobase (kb) to a few hundred kb segments, and contain an origin of replication which allows the plasmid to replicate independently of the bacterial chromosome.  The total number of a particular plasmid within a cell is referred to as the copy number and can range from as few as two copies per cell to as many as several hundred copies per cell. Mitochondrial DNA  are a main source of this extrachromosomal DNA in eukaryotes .

4. Extrachromosomal  DNA in the cytoplasm have been found to be structurally different from nuclear DNA.  Cytoplasmic DNA are less  methylated  than DNA found within the nucleus. In addition to DNA found outside the nucleus in cells, infection of viral genomes also provides an example of  extrachromosomal DNA. Extrachromosomal DNA are often used in research of replication because they are easy to identify and isolate.

5. PLASMID Plasmids are small, circular, double stranded, few kilo base self-replicating extra DNA fragments commonly recognized in different gram negative and positive bacterial strains as well as in some fungi including unicellular  yeasts  They are capable of self replication independent of the host genome.  Though plasmids are not required for survival of a living organism it encodes essential genetic determinants that enable an organism to adapt and resist unfavourable conditions for better survival . Most of them are covalently closed circular double-stranded DNA molecules, recently linear plasmids have been isolated from different bacteria. Their existence was initially revealed as the "F factor" in  Escherichia coli  even before the double-helix structure of DNA was elucidated by Watson and Crick.

6. REPLICATION Several host- and plasmid-encoded factors are required for plasmid replication.   Plasmid replicons consists of one or more  origin of replication ( ori )  and few regulatory elements such as  Rep proteins , localized in the 4 kilo base region of the DNA fragment.  In addition, most plasmid replicons harbor a   gene encoding either a protein or an RNA molecule that functions as a primer for DNA replication . It was reported that many plasmid origins follow a molecular mechanism similar to  ori C , the origin of replication of the  E. coli  chromosome. However, the major difference is that plasmids require an origin-specific plasmid-encoded protein for the initiation step, generally called Rep proteins . Rolling circle, Col E1 type  and  iteron-containing replicons  are the common modes through which plasmid replicates, each mechanism with unique significance to the organism.  

7. Mainly three mechanism; 1. Rolling circle mechanism 2.Iteron regulated mechanism 3.RNA regulated-Col E1 mechanism

8. Rolling Circle Mechanism   The most common replication system among the Gram-positives plasmids is the rolling circle. Rolling circle replication mechanism is specific to bacteriophage family m13 and the fertility F factor which encodes for sex pili formation during recombination by means of conjugation.   Fragments smaller than 10 kilo base usually replicate by this replication mechanism as reported in some gram positive bacteria. It allows the transfer of single stranded replication product at a faster rate to the recipient cell through pilus as in case of fertility factor or to the membrane in case of phage.

9. Rolling circle DNA replication is initiated by an initiator protein ( RepA ) encoded by the plasmid or bacteriophage DNA, which nicks one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin, or DSO. The initiator protein remains bound to the 5' phosphate end of the nicked strand, and the free 3' hydroxyl end is released to serve as a primer for DNA synthesis by host DNA polymerase III. Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA. Displacement (unwinding) of the nicked strand is carried out by a host-encoded helicase called PcrA (the abbreviation standing for plasmid copy reduced) in the presence of RepA .

10. As the DNA unwinds it becomes coded by single-strand DNA binding proteins . As replication proceeds the nick strand which continues to be covered with single-strand DNA binding proteins progressively peels off until replication of the intact strand is complete. The two ends of the nicked single strand are rejoined by the RepA  protein and released. DNA ligase seals the nick in the double stranded molecule. The single-strand DNA can now be replicated. A region of the DNA becomes looped, allowing RNA polymerase access to DNA to form a primer.

11. Host DNA polymerases use the primer as a starting point for the synthesis of DNA. RNA polymerase  and DNA polymerase III then replicate the single-stranded origin (SSO) DNA to make another double-stranded circle. DNA polymerase I removes the primer, replacing it with DNA, and  DNA ligase  joins the ends to make another molecule of double-stranded circular DNA. Each of these plasmids can undergo replication again by the same method.

12. Iteron -regulated plasmid s Iterons are directly repeated DNA sequences which play an important role in regulation of plasmid copy number in bacterial cells. Iterons complex with cognate replication (Rep) initiator proteins to achieve the required regulatory effect. This replicon consists of :  a gene that encodes Rep protein ( replication initiator protein ) for plasmid replication initiation Origin of replication ( ori ) set of direct repeat sequences called iteron (located within the ori ) adjacent AT-rich region   DnaA boxes which is a protein required for bacterial chromosome replication initiation.  Iterons are the primary DNA binding sites for Rep protein and these sequences arranged in tandem, direct repeats. (DR ) The conjugative plasmid R6K, a naturally occurring extrachromosomal element that codes for resistance to the antibiotics ampicillin and streptomycin  belongs to the group of iteron-regulated replicons. It is about 38 kb in size and has a copy number of 13 to 40 per cell .

13. Mechanism : Iteron contain replication begins with the binding of Rep proteins to the iteron being organized in the same orientation of the DNA helix. Then this  binds to the DnaA boxes in the replicon, the Rep- DnaA -DNA assembly promotes melting of the strand at the nearby AT-rich region to which host replication factors subsequently gain access and promote leading and lagging strand synthesis in a manner analogous to initiation of replication at the chromosomal origin, oriC .

14. Representation of iterons

15. Replication Initiator Proteins The replication initiator protein (Rep) plays a key role in initiation of replication in plasmids. In its monomer form, Rep binds an iteron and promotes replication. The protein itself is known to contain two independent N-terminal and C-terminal globular domains that subsequently bind to two domains of the iteron. The dimer version of the protein is generally inactive in iteron binding, however it is known to bind to the repE  operator. This operator contains half of the iteron sequence making it able to bind the dimer and preventing gene expression. Plasmids containing iterons are all organized very similarly in structure. The gene for Rep proteins is usually found directly downstream of the origin of replication. This means that the iterons themselves are known to regulate the synthesis of the rep proteins.

16. Iterons have an important role in plasmid replication. An iteron-containing plasmid origin of replication can be found containing about five iterons about 20 base pairs in length total. These iterons provide a saturation site for initiator receptor proteins and promote replication thus increasing plasmid copy number in a given cell.

17. RNA regulated – Col E1 type Col E1 is a plasmid found in bacteria.  Col E1 replication is a negative regulation mechanism which enables the plasmid to control its own copy numbers by involving RNA I , RNA type II, Rom protein, and the plasmid itself. Col E1 replication is initiated by means of RNA-RNA interactions and does not rely on replication initiation protein encoded by the plasmid to regulate its copy number. Mechanism : RNA polymerase initiates transcription of RNA II that originates 555 base pairs upstream from the replication origin of Col E1 plasmid which marks the start of Col E1 replication. A determined hybrid with the DNA strand is formed by a loop enriched in G nucleotide of RNAII and a C-rich region on the template strand upstream from the origin. The transcript folds into a secondary structure which stabilises the interaction between the nascent RNA and the origin's DNA. Several stems and loops are exhibited by the newly formed secondary structure.

18. This hybrid is attacked by RNase H , which digests the RNA II at the replication origin, on recognizing this RNA II-DNA duplex.  As a consequence a free 3'-hydroxyl group is generated that serves as primer for DNA synthesis catalyzed by DNA polymerase 1 . Once DNA polymerase 1 begins the addition of deoxynucleotides, the remaining portion of RNA II which is still hybridized to the template DNA is digested at other sites by RNase H and by the 5'-3' exonuclease activity of DNA polymerase 1.  ColE1 DNA replication proceeds unidirectionally with the initiation of the lagging strand synthesis at specific ColE1 sites.  Replication is carried out entirely by host proteins (RNA polymerase, DNA polymerase I and RNase H) so that inhibition of translation will stop the growth of the cells, but not the replication of ColE1. Since the translation of Rop protein will also be inhibited, a higher than normal copy number will result in these cells.

19. RNAI is a counter-transcript to a section of RNAII and so binds to its 5' end. This alters the folding of RNAII so that the DNA-RNA hybrid is not stabilized and cleavage does not occur. This ensures that at high copy numbers, replication is slowed down due to increased RNAI concentration. Rop  is a secondary replication repressor, it stabilizes the RNAI-RNAII hybrid. Rop may be especially important at preventing runaway replication, at slow growth rates.

20. COPY NUMBER AND PARTITIONING Copy number refers to the average or expected number of copies per host cell. Plasmids are either low, medium or high copy number. Plasmids vary widely in copy number depending on three main factors: 1) The  ori  and its constituents – (e.g. ColE1 RNA I and RNA II). 2) The size of the plasmid and its associated insert (bigger inserts and plasmids may be replicated at a lower number as they represent a great metabolic burden for the cell). 3) Culture conditions (i.e. factors that influence the metabolic burden on the host).

21. Partition is generally the most important determinant of the stability of low-copy-number plasmids, which are common in bacteria. In contrast, high-copy-number plasmids typically do not encode partition systems because random segregation is sufficient for stability.  Partition is a dynamic process; plasmids are moved and positioned inside the cell so that cell division separates at least one copy into each daughter cell.  Random segregation of low-copy-number plasmids (only 2 to 4 copies per cell) would likely mean that, following cell division, one of the daughter cells would not receive a plasmid. The plasmid would eventually be diluted from the population. Consequently, regulated partitioning mechanisms are essential for these plasmids. 

22. MITOCHONDRIAL DNA REPLICATION Mammalian mitochondria contain multiple copies of a circular, double-stranded DNA genome. Mitochondrial DNA ( mtDNA ) is a double-stranded molecule of 16.6 kb . Mammalian   mtDNA  is replicated by proteins distinct from those used for nuclear DNA replication and many are related to replication factors identified in bacteriophages.

23.   The two strands of  mtDNA  differ in their base composition, with one being rich in guanines, making it possible to separate a  heavy (H)  and a  light (L) strand  by density centrifugation in alkaline CsCl2 gradients.  The  mtDNA  contains one longer  noncoding region (NCR)  also referred to as the  control region .D loop  In the NCR, there are promoters for polycistronic transcription, one for each  mtDNA  strand; the  light strand promoter (LSP)  and the  heavy strand promoter (HSP) .  The NCR also harbors the  origin for H-strand DNA replication (OH).   A minor NCR, located approximately 11,000 bp downstream of O H , contains the second  origin for L-strand DNA replication (OL).

24. mtDNA  REPLICATION FACTORS DNA polymerase γ ( POLγ ) is the replicative polymerase in mitochondria . In human cells, POLγ is a heterotrimer with one catalytic subunit ( POLγA ) and two accessory subunits ( POLγB ). The DNA helicase TWINKLE is homologous to the T7 phage gene 4 protein and during mtDNA replication, TWINKLE travels in front of POLγ , unwinding the double-stranded DNA template . TWINKLE forms a hexamer and requires a fork structure (a single-stranded 5′-DNA loading site and a short 3′-tail) to load and initiate unwinding . Mitochondrial single-stranded DNA-binding protein ( mtSSB ) binds to the formed ssDNA, protects it against nucleases, and prevents secondary structure formation .

25. The model of mtDNA replication A model for mtDNA replication was presented already in 1972 by Vinograd and co-workers.   According to their strand displacement model , DNA synthesis is continuous on both the H- and L-strand.  There is a dedicated origin for each strand, O H  and O L . First, replication is initiated at O H  and DNA synthesis then proceeds to produce a new H-strand.  During the initial phase, there is no simultaneous L-strand synthesis and mtSSB covers the displaced, parental H-strand.  By binding to single-stranded DNA, mtSSB prevents the mitochondrial RNA polymerase (POLRMT) from initiating random RNA synthesis on the displaced strand. 

26. When the replication fork has progressed about two-thirds of the genome, it passes the second origin of replication, O L .  When exposed in its single-stranded conformation, the parental H-strand at O L  folds into a stem–loop structure .  The stem efficiently blocks mtSSB from binding and a short stretch of single-stranded DNA in the loop region therefore remains accessible, allowing POLRMT to initiate RNA synthesis. POLRMT is not processive on a single-stranded DNA templates. Already after about 25 nt , it is replaced by POLγ and L-strand DNA synthesis is initiated. From this point, H- and L-strand synthesis proceeds continuously until the two strands have reached full circle. Replication of the two strands is linked , since H-strand synthesis is required for initiation of L-strand synthesis. 

27. When POLγ has completed synthesis, the newly formed DNA strands are ligated by DNA ligase III. To allow for efficient ligation , the RNA primers used to initiate mtDNA synthesis must first be removed. A likely candidate for primer removal is RNASEH1 . After completing a full circle-replication, POLγ encounters the 5′-end of the nascent full-length mtDNA strand it has just produced.  At this point, POLγ initiates successive cycles of polymerization and 3′–5′ exonuclease degradation at the nick. This process, idling, is required for proper ligation ..

28. SEPARATION mtDNA During DNA replication, the parental molecule remains intact, which poses a steric problem for the moving replication machinery. Topoisomerases belonging to the type 1 family can relieve torsional strain formed in this way, by allowing one of the strands to pass through the other. In mammalian mitochondria, TOP1MT a type IB enzyme can act as a DNA “swivel ”. R eplication of intact, circular DNA generates daughter molecules linked together as  catenanes , i.e. mechanically interlocked, but not yet completely finished DNA circles. Therefore , replication of circular genomes requires decatenation to generate complete daughter molecules separation. .

29. The existence of catenanes in mitochondria was reported already in 1967 by Vinograd and co-workers, who identified mtDNA molecules linked together during completion of mtDNA replication. Recently, it was demonstrated that these s tructures are hemicatenanes , i.e. double-stranded DNA molecules linked together via a single-stranded linkage   A mitochondrial isoform of Topoisomerase 3α (Top3α) is required to resolve the hemicatenane structure

30. CHLOROPLASTS Chloroplasts contain multiple copies of a DNA molecule(the plastome ) that encodes many of the gene products required to perform photosynthesis.  The plastome is replicated by nuclear-encoded proteins and its copy number seems to be highly regulated by the cell in a tissue-specific and developmental manner. The chloroplast genome ( plastome ) exists as a covalently closed, double stranded circular ranging in size from 120 kilobase pairs ( kbp ) in some species to over 200  kbp  in others.

31. ctDNA REPLICATION The currently prevailing model of DNA replication in chloroplasts is based on electron microscopic examination of replication intermediates in isolated pea and maize  ctDNA , and was put forth by Kolodner and  Tewari . Replication begins at two displacement (D-) loop  initiation sites located on opposite strands approximately 7  kbp  apart.  The D-loops expand unidirectionally toward each other until the advancing forks pass the D-loop initiation site on the opposite strand, at which point discontinuous replication begins, resulting in two Cairns-type, bidirectional forks moving away from each other .  Presumably the forks meet at some point approximately  180 degrees from the starting point and give rise to two daughter molecules.

32. Unidirectional elongation of nascent strand initiated from both origins. Unidirectional fork movement toward each other. Fusion of D-loops. Bidirectional, semi-discontinuous replication. Resulting daughter molecules. 

33. Replication enzymes : These include origin recognition protein(s ), DNA unwinding activity ( helicase ), single stranded DNA binding protein topoisomerase I and II, single stranded DNA binding protein DNA polymerase(s), Results obtained with diverse plant types and with  plastomes  of different morphologies do not yet allow us to present a generalized picture of  ctDNA  replication. What can be stated with confidence is that in higher plants the  ctDNA  replication machinery seems to be encoded in the nucleus, which would place regulation of  plastome  synthesis under cellular control.

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