Chapters 16, 15 & 17 Genetic Recombination & DNA Repair • Recombination occurs in a manner which prevents the loss or insertion of DNA bases. Three types of recombination:

– Homologous recombination – also known as generalized; occurs at meiosis, (as we have previously discussed)

– Site specific (specialized) recombination – typically in bacteria and viruses; involved act only on a particular pair of target sequences in an intermolecular reaction

– Transposition (including gene rearrangements) – where DNA sequences can be inserted into another sequence without relying on sequence homology between the two. DNA Transposition Example (s)

Replicative Tn3 Transposase / Resolvase

Nonreplicative two-strand Tn5 Transposase

Tn7 Transposase (TnsB) + Endonuclease (TnsA)

Nonreplicative four-strand Tn10 Transposase

Ac Transposase autonomous (controlled by Methylation) (Ds) nonautonomous

3 DNA Transposition Example Enzyme(s)

Replicative Tn3 Transposase / Resolvase

Nonreplicative two-strand Tn5 Transposase

Tn7 Transposase (TnsB) + Endonuclease (TnsA)

Nonreplicative four-strand Tn10 Transposase

Ac Transposase autonomous (controlled by Methylation) (Ds) nonautonomous

4 DNA Transposition Example Enzyme(s)

Replicative Tn3 Transposase / Resolvase

Nonreplicative two-strand Tn5 Transposase

Tn7 Transposase (TnsB) + Endonuclease (TnsA)

Nonreplicative four-strand Tn10 Transposase

Ac Transposase autonomous (controlled by Methylation) (Ds) nonautonomous

5 DNA Transposition Example Enzyme(s)

Replicative Tn3 Transposase / Resolvase Transposase (TnsB) Tn7 - Endonuclease (TnsA)

Nonreplicative two-strand Tn5 Transposase

Tn7 Transposase (TnsB) + Endonuclease (TnsA)

Nonreplicative four-strand Tn10 Transposase

Ac Transposase autonomous (controlled by Methylation) (Ds) nonautonomous

6 DNA Transposition Example Enzyme(s)

Replicative Tn3 Transposase / Resolvase

Nonreplicative two-strand Tn5 Transposase

Tn7 Transposase (TnsB) + Endonuclease (TnsA)

Nonreplicative four-strand Tn10 Transposase

Ac Transposase autonomous (controlled by Methylation) (Ds) nonautonomous

7 DNA Transposition Example Enzyme(s)

Replicative Tn3 Transposase / Resolvase

Nonreplicative two-strand Tn5 Transposase

Tn7 Transposase (TnsB) + Endonuclease (TnsA)

Nonreplicative four-strand Tn10 Transposase

Ac Transposase autonomous (controlled by Methylation) (Ds) nonautonomous

8 DNA Transposition Example Enzyme(s)

Replicative Tn3 Transposase / Resolvase

Nonreplicative two-strand Tn5 Transposase

Tn7 Transposase (TnsB) + Endonuclease (TnsA)

Nonreplicative four-strand Tn10 Transposase

Ac Transposase autonomous (controlled by Methylation) (Ds) nonautonomous

9 • Recombination occurs in a manner which prevents the loss or insertion of DNA bases. Three types of recombination:

– Homologous recombination – also known as generalized; occurs at meiosis, (as we have previously discussed)

– Site specific (specialized) recombination – typically in bacteria and viruses; enzymes involved act only on a particular pair of target sequences in an intermolecular reaction

– Transposition (including gene rearrangements) – where DNA sequences can be inserted into another sequence without relying on sequence homology between the two. Figure 1. Models for the repair of DNA double- strand breaks. DNA DSBs are resected to generate 3’-protruding ends followed by formation of Rad51 filaments that invade into homologous template to form D-loop structures.

(A)After priming DNA synthesis, three pathways can be invoked. In the DSBR pathway, the second end is captured and a dHJ intermediate is formed.

(B) Resolution of dHJs can occur in either plane to generate crossover or non-crossover products. Alternatively, dHJs can be dissolved by the action of Sgs1–Top1–Rmi1 complex to generate primarily non-crossovers. In the SDSA pathway

(C) the extended nascent strand is displaced, followed by pairing with the other 3’-single- stranded tail, and DNA synthesis completes repair. Nucleolytic trimming might be also required.

(D)In the third pathway of BIR which can act when the second end is absent, the D-loop intermediate turns into a replication fork capable of both lagging and leading strand synthesis.

(E) Two other Rad51-independent recom- binational repair pathways are also depicted. In SSA extensive resection can reveal complementary sequences at two repeats, allowing annealing. The 3’-tails are removed nucleolytically and the nicks are ligated. SSA leads to the deletion of one of the repeats and the intervening DNA.

(F) Finally, the ends of DSB can be directly ligated resulting in NHEJ. Newly synthesized DNA is represented by dashed lines. Figure 1. Models for the repair of DNA double- strand breaks. DNA DSBs are resected to generate 3’-protruding ends followed by formation of Rad51 filaments that invade into homologous template to form D-loop structures.

(A)After priming DNA synthesis, three pathways can be invoked. In the DSBR pathway, the second end is captured and a dHJ intermediate is formed.

(B) Resolution of dHJs can occur in either plane to generate crossover or non-crossover products. Alternatively, dHJs can be dissolved by the action of Sgs1–Top1–Rmi1 complex to generate primarily non-crossovers. In the SDSA pathway

(C) the extended nascent strand is displaced, followed by pairing with the other 3’-single- stranded tail, and DNA synthesis completes repair. Nucleolytic trimming might be also required.

(D)In the third pathway of BIR which can act when the second end is absent, the D-loop intermediate turns into a replication fork capable of both lagging and leading strand synthesis.

(E) Two other Rad51-independent recom- binational repair pathways are also depicted. In SSA extensive resection can reveal complementary sequences at two repeats, allowing annealing. The 3’-tails are removed nucleolytically and the nicks are ligated. SSA leads to the deletion of one of the repeats and the intervening DNA.

(F) Finally, the ends of DSB can be directly ligated resulting in NHEJ. Newly synthesized DNA is represented by dashed lines. Creighton, H., and McClintock, B. 1931 A correlation of cytological and genetical crossing-over in Zea mays. PNAS 17:492–497. Break Induced Replication (BIR) initiates translocations Break Induced Recombination (BIR) initiating translocations, sometimes through regional homology with DNA potentially from other

15 https://www.youtube.com/watch?v=31stiofJjYw Creighton, H., and McClintock, B. 1931 A correlation of cytological and genetical crossing-over in Zea mays. PNAS 17:492–497.

A break at a “controlling element” often also caused the loss of an acentric fragment on this ; if the fragment carried the dominant markers of a heterozygote, its loss would change the phenotype. • provided one of the earliest models of transposition in eukayotes, which was discovered because of the effects of chromosome breaks that were generated by transposition of “controlling elements”. The breaks generate on one chromosome that has a centromere, a broken end, and one acentric fragment.

• In maize, transposable elements often insert near genes that have visible but non-lethal effects on the phenotype. • Maize displays clonal development which means that the occurrence and timing of a transposition event can be visualized as the resulting progeny following a “break” give rise to a sector – or patch of cells made up of a single altered cell and its progeny. Ac-im contributes to negative dosage.

Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize

FIGURE 1.— Ac-im contributes to negative dosage. Aleurone variegation patterns of ears carrying (A) one, (B) two, or (C) three copies of Ac-im in the triploid endosperm. Kernels with fully colored aleurone (clearly visible in B and C) carry revertant R1-sc alleles resulting from excisions of the Ds at r1.

Conrad L J , Brutnell T P Genetics 2005;171:1999-2012

Copyright © 2005 by the Genetics Society of America Ac-im contributes to negative dosage.

Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize

FIGURE 1.— Ac-im contributes to negative dosage. Aleurone variegation patterns of ears carrying (A) one, (B) two, or (C) three copies of Ac-im in the triploid endosperm. Kernels with fully colored aleurone (clearly visible in B and C) carry revertant R1-sc alleles resulting from excisions of the Ds at r1.

Conrad L J , Brutnell T P Genetics 2005;171:1999-2012

Copyright © 2005 by the Genetics Society of America Ac-im contributes to negative dosage.

Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize

FIGURE 1.— Ac-im contributes to negative dosage. Aleurone variegation patterns of ears carrying (A) one, (B) two, or (C) three copies of Ac-im in the triploid endosperm. Kernels with fully colored aleurone (clearly visible in B and C) carry revertant R1-sc alleles resulting from excisions of the Ds at r1.

Conrad L J , Brutnell T P Genetics 2005;171:1999-2012

Copyright © 2005 by the Genetics Society of America Ac-im contributes to negative dosage.

Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize

FIGURE 1.— Ac-im contributes to negative dosage. Aleurone variegation patterns of ears carrying (A) one, (B) two, or (C) three copies of Ac-im in the triploid endosperm. Kernels with fully colored aleurone (clearly visible in B and C) carry revertant R1-sc alleles resulting from excisions of the Ds at r1.

Conrad L J , Brutnell T P Genetics 2005;171:1999-2012

Copyright © 2005 by the Genetics Society of America The C locus produces an anthocyanin pigment, resulting in a coloured aleurone ie. wild-type.

Insertion of a transposon into C blocks pigment production, resulting in the colorless (c) phenotype.

During seed development, excision of the Ds element can result in patches of cells (each “mitotic” progeny of a single cell) expressing a colored phenotype. This is referred to as variegation.

If the Ac locus moves to a position adjacent to Ds, where it is now able to promotes the movement of Ds away from the C locus during the development of the kernel -and the locus reverts to normal expression.

The larger the number of Ac factors present, the greater expression of variegation in the tissue.

Larger coloured patches are formed when Ds is expressed early in development; smaller patches when Ds is expressed late. 26 • The fusion-breakage-bridge cycle is also potentially responsible for the occurrence of somatic variegation.

27 Transposons move from one place to another within the genetic material of Indian corn. When the transposon moves into the pigment gene, it stops pigments production and the Indian corn kernels appears white. When the transposon moves out, pigment is produced again. So, this variable in and out movement of transposons determines the colors of the Indian corn mosaic. 28 Maize elements form families of transposons that can be autonomous or nonautonomous. • Autonomous elements encode the transposase and have the ability to excise and transpose; insertion of such an element creates an unstable allele while the loss of the element converts a mutable allele into a stable one. • Nonautonomous elements are relatively stable and do not transpose as they cannot catalyze transposition, but they can transpose when an autonomous element provides the necessary proteins. They are derived from autonomous elements by loss of the transacting functions needed for transposition. • Autonomous elements are subject to changes of phase, which are heritable but relatively unstable alterations in their properties. In the Ac and Mu types of Ac element – Activator element; an autonomous in maize. elements, these phases are regulated by Ds element – Dissociation element; a nonautonomous methylation state of DNA. transposable element in maize, related to the autonomous Activator (Ac) element. Maize elements form families of transposons that can be autonomous or nonautonomous. • Autonomous elements encode the transposase and have the ability to excise and transpose; insertion of such an element creates an unstable allele while the loss of the element converts a mutable allele into a stable one. • Nonautonomous elements are relatively stable and do not transpose as they cannot catalyze transposition, but they can transpose when an autonomous element provides the necessary proteins. They are derived from autonomous elements by loss of the transacting functions needed for transposition. • Autonomous elements are subject to changes of phase, which are heritable but relatively unstable alterations in their properties. In the Ac and Mu types of Ac element – Activator element; an autonomous transposable element in maize. elements, these phases are regulated by Ds element – Dissociation element; a nonautonomous methylation state of DNA. transposable element in maize, related to the autonomous Activator (Ac) element. Maize elements form families of transposons that can be autonomous or nonautonomous. • Autonomous elements encode the transposase and have the ability to excise and transpose; insertion of such an element creates an unstable allele while the loss of the element converts a mutable allele into a stable one. • Nonautonomous elements are relatively stable and do not transpose as they cannot catalyze transposition, but they can transpose when an autonomous element provides the necessary proteins. They are derived from autonomous elements by loss of the transacting functions needed for transposition. • Autonomous elements are subject to changes of phase, which are heritable but relatively unstable alterations in their properties. In the Ac and Mu types of Ac element – Activator element; an autonomous transposable element in maize. elements, these phases are regulated by Ds element – Dissociation element; a nonautonomous methylation state of DNA. transposable element in maize, related to the autonomous Activator (Ac) element. Maize elements form families of transposons that can be autonomous or nonautonomous. • Autonomous elements encode the transposase and have the ability to excise and transpose; insertion of such an element creates an unstable allele while the loss of the element converts a mutable allele into a stable one. • Nonautonomous elements are relatively stable and do not transpose as they cannot catalyze transposition, but they can transpose when an autonomous element provides the necessary proteins. They are derived from autonomous elements by loss of the transacting functions needed for transposition. • Autonomous elements are subject to changes of phase, which are heritable but relatively unstable alterations in their properties. In the Ac and Mu types of Ac element – Activator element; an autonomous transposable element in maize. elements, these phases are regulated by Ds element – Dissociation element; a nonautonomous methylation state of DNA. transposable element in maize, related to the autonomous Activator (Ac) element. Maize elements form families of transposons that can be autonomous or nonautonomous. • Autonomous elements encode the transposase and have the ability to excise and transpose; insertion of such an element creates an unstable allele while the loss of the element converts a mutable allele into a stable one. • Nonautonomous elements are relatively stable and do not transpose as they cannot catalyze transposition, but they can transpose when an autonomous element provides the necessary proteins. They are derived from autonomous elements by loss of the transacting functions needed for transposition. • Autonomous elements are subject to changes of phase, which are heritable but relatively unstable alterations in their properties. In the Ac and Mu types of Ac element – Activator element; an autonomous transposable element in maize. elements, these phases are regulated by Ds element – Dissociation element; a nonautonomous methylation state of DNA. transposable element in maize, related to the autonomous Activator (Ac) element. Maize elements form families of transposons that can be autonomous or nonautonomous. • Autonomous elements encode the transposase and have the ability to excise and transpose; insertion of such an element creates an unstable allele while the loss of the element converts a mutable allele into a stable one. • Nonautonomous elements are relatively stable and do not transpose as they cannot catalyze transposition, but they can transpose when an autonomous element provides the necessary proteins. They are derived from autonomous elements by loss of the transacting functions needed for transposition. • Autonomous elements are subject to changes of phase, which are heritable but relatively unstable alterations in their properties. In the Ac and Mu types of Ac element – Activator element; an autonomous transposable element in maize. elements, these phases are regulated by Ds element – Dissociation element; a nonautonomous methylation state of DNA. transposable element in maize, related to the autonomous Activator (Ac) element. Maize elements form families of transposons that can be autonomous or nonautonomous. • Autonomous elements encode the transposase and have the ability to excise and transpose; insertion of such an element creates an unstable allele while the loss of the element converts a mutable allele into a stable one. • Nonautonomous elements are relatively stable and do not transpose as they cannot catalyze transposition, but they can transpose when an autonomous element provides the necessary proteins. They are derived from autonomous elements by loss of the transacting functions needed for transposition. • Autonomous elements are subject to changes of phase, which are heritable but relatively unstable alterations in their properties. In the Ac and Mu types of Ac element – Activator element; an autonomous transposable element in maize. elements, these phases are regulated by Ds element – Dissociation element; a nonautonomous methylation state of DNA. transposable element in maize, related to the autonomous Activator (Ac) element. Maize elements form families of transposons that can be autonomous or nonautonomous. • Autonomous elements encode the transposase and have the ability to excise and transpose; insertion of such an element creates an unstable allele while the loss of the element converts a mutable allele into a stable one. • Nonautonomous elements are relatively stable and do not transpose as they cannot catalyze transposition, but they can transpose when an autonomous element provides the necessary proteins. They are derived from autonomous elements by loss of the transacting functions needed for transposition. • Autonomous elements are subject to changes of phase, which are heritable but relatively unstable alterations in their properties. In the Ac and Mu types of Ac element – Activator element; an autonomous transposable element in maize. elements, these phases are regulated by Ds element – Dissociation element; a nonautonomous methylation state of DNA. transposable element in maize, related to the autonomous Activator (Ac) element. Ds elements in maize arise by deletions of Ac

37 Transposons related to the Spm family of elements are found in a variety of plants. They all share nearly identical inverted terminal repeats and generate 3 bp duplications of target DNA upon transposition. They are also known as the CACTA group of transposons.

• The Spm element includes the tnpA gene product (which is required for excision but may not be sufficient for transposition) and the tnpB product thought to bind to the 13 bp inverted repeats to cleave the termini for transposition

• Spm insertions can control the expression of a gene at the site of insertion. • P elements and hybrid dysgenesis (hybrid deficiencies). P elements are found in 30-40 copies of certain Drosophila . They come in various sizes and modifications, but all contain a 31 bp perfect, terminal inverted repeats. Essentially there are two types of Drosophila populations M and P types (i.e. those that have the [P] and those that haven't [M]). • Moreover, crossing these two strains give conflicting genetics…. • Cross any Male P with a Female M and ...... all hell (heck!) breaks loose, leading to… • Hybrid dysgenesis, – The inability of certain strains of D. melanogaster to interbreed, because the hybrids are sterile (although otherwise they may be phenotypically normal). • with respect to P elements this was first determined when "accidentally" pure bread Drosophila strains were mated with Drosophila from the wild. • The resulting, “anomolous genetics” was hard to appreciate, at first.

P elements are transposons that are carried in P strains of Drosophila melanogaster.

Transposition is activated when a P male is crossed with an M female.

Yet, M male is crossed with P female... nothing anomolous happens

Any one chromosome of the P male can induce the hybrid dysgenesis effect. 39 The P element has four exons.

• The P strain carries 30 to 50 copies of the P element, which becomes “activated” when a P male is crossed with a “naive” M female.

• The Tn insertion inactivates genes in which they are located, and often causes chromosomal breaks…. leading to sterility.

• This activation occurs because a tissue-specific splicing event, that occurs in the germ-line cells, gives rise to the removal of intron 3 in a P elemental gene, which generates an 87 kD protein, which functions as a transposase. P Elements Are Activated in the Germline

• The P element also produces a repressor of transposition, which is inherited maternally in the cytoplasm.

• The presence of the repressor explains why M male × P female crosses remain fertile.

• Hybrid dysgenesis is determined by the interacons between P elements in the and repressors in the cytotype. Different types or “nucleotide”

Dd Dp DNA -dependent DNA polymerases ? DNA replicases prokaryotes /eukaryotes DNA repair enzymes

Dd Rp DNA -dependent RNA polymerases ?

RNA polymerases -transcription

Rd Dp RNA -dependent DNA polymerases ?

Telomerase

42 Different types or “nucleotide” polymerases

Dd Dp DNA -dependent DNA polymerases ? DNA replicases prokaryotes /eukaryotes DNA polymerase repair enzymes

Dd Rp DNA -dependent RNA polymerases ?

RNA polymerases -transcription

Rd Dp RNA -dependent DNA polymerases ?

Telomerase

43 Different types or “nucleotide” polymerases

Dd Dp DNA -dependent DNA polymerases ? DNA replicases prokaryotes /eukaryotes DNA polymerase repair enzymes

Dd Rp DNA -dependent RNA polymerases ?

RNA polymerases -transcription

Rd Dp RNA -dependent DNA polymerases ?

Telomerase

Rd Rp RNA -dependent RNA polymerases ?

44 45 46 47 Overview of transcription and replication strategies for various types of RNA viruses. (a) Positive-strand RNA [(+)RNA] viruses. The 40 genomes of (+)RNA viruses are message-sense (green), and they often contain a 5ʹ m7G cap (purple circle) and 3ʹ poly-A tail (AAAAA). Host cell 41 ribosomes translate the genome into one or more polyproteins, which are cotranslationally and posttranslationally processed by virally encoded 42 proteases. Some of the mature polyprotein processing precursors and products include the RNA- dependent RNA polymerase (RdRp; light blue 43 rectangle) and cofactors (light blue squares) that mediate viral RNA synthesis in association with cellular membranes. Other proteins made by the 44 virus include those that will assemble into viral particles (gray squares). The RdRp mediates the synthesis of negative- strand RNA [(−)RNA] 45 antigenome (red) using the genome as template. The antigenome is then converted into new (+)RNA genome by the RdRp and then packaged into 46 nascent virion particles (gray hexagon). (b) Negative- strand RNA [(−)RNA] viruses. The genomes of (−)RNA viruses are organized as 47 ribonucleoproteins (RNPs) with their RNA molecules (red) bound by the viral polymerase complex (light blue rectangle) and nucleocapsid proteins (N; blue square). 48 Transcription is mediated by the RdRp and proteins, resulting in the synthesis of 5ʹ capped (purple circle), 3ʹ polyadenylated (AAAAA) mRNAs (green). The mRNAs are Overview of transcription and replication strategies for various types of RNA viruses. (a) Positive-strand RNA [(+)RNA] viruses. The 40 genomes of (+)RNA viruses are message-sense (green), and they often contain a 5ʹ m7G cap (purple circle) and 3ʹ poly-A tail (AAAAA). Host cell 41 ribosomes translate the genome into one or more polyproteins, which are cotranslationally and posttranslationally processed by virally encoded 42 proteases. Some of the mature polyprotein processing precursors and products include the RNA- dependent RNA polymerase (RdRp; light blue 43 rectangle) and cofactors (light blue squares) that mediate viral RNA synthesis in association with cellular membranes. Other proteins made by the 44 virus include those that will assemble into viral particles (gray squares). The RdRp mediates the synthesis of negative- strand RNA [(−)RNA] 45 antigenome (red) using the genome as template. The antigenome is then converted into new (+)RNA genome by the RdRp and then packaged into 46 nascent virion particles (gray hexagon). (b) Negative- strand RNA [(−)RNA] viruses. The genomes of (−)RNA viruses are organized as 47 ribonucleoproteins (RNPs) with their RNA molecules (red) bound by the viral polymerase complex (light blue rectangle) and nucleocapsid proteins (N; blue square). 48 Transcription is mediated by the RdRp and cofactor proteins, resulting in the synthesis of 5ʹ capped (purple circle), 3ʹ polyadenylated (AAAAA) mRNAs (green). The mRNAs are Overview of transcription and replication strategies for various types of RNA viruses. (a) Positive-strand RNA [(+)RNA] viruses. The 40 genomes of (+)RNA viruses are message-sense (green), and they often contain a 5ʹ m7G cap (purple circle) and 3ʹ poly-A tail (AAAAA). Host cell 41 ribosomes translate the genome into one or more polyproteins, which are cotranslationally and posttranslationally processed by virally encoded 42 proteases. Some of the mature polyprotein processing precursors and products include the RNA- dependent RNA polymerase (RdRp; light blue 43 rectangle) and cofactors (light blue squares) that mediate viral RNA synthesis in association with cellular membranes. Other proteins made by the 44 virus include those that will assemble into viral particles (gray squares). The RdRp mediates the synthesis of negative- strand RNA [(−)RNA] 45 antigenome (red) using the genome as template. The antigenome is then converted into new (+)RNA genome by the RdRp and then packaged into 46 nascent virion particles (gray hexagon). (b) Negative- strand RNA [(−)RNA] viruses. The genomes of (−)RNA viruses are organized as 47 ribonucleoproteins (RNPs) with their RNA molecules (red) bound by the viral polymerase complex (light blue rectangle) and nucleocapsid proteins (N; blue square). 48 Transcription is mediated by the RdRp and cofactor proteins, resulting in the synthesis of 5ʹ capped (purple circle), 3ʹ polyadenylated (AAAAA) mRNAs (green). The mRNAs are Overview of transcription and replication strategies for various types of RNA viruses. (a) Positive-strand RNA [(+)RNA] viruses. The 40 genomes of (+)RNA viruses are message-sense (green), and they often contain a 5ʹ m7G cap (purple circle) and 3ʹ poly-A tail (AAAAA). Host cell 41 ribosomes translate the genome Overview of transcription and replication strategiesinto one for or more various polyproteins, types whichof (a) are Positive-strand RNA [(+)RNA] viruses. cotranslationally and posttranslationally processed by virally encoded 42 proteases. Some of the mature polyprotein processing ʹ 7 The genomes of (+)RNA viruses are message-senseprecursors (green), and products and includethey oftenthe RNA- contain a 5 m G cap (purple circle) and 3ʹ poly-A tail (AAAAA). dependent RNA polymerase (RdRp; light blue 43 rectangle) and cofactors (light blue squares) Host cell ribosomes translate the genome into onethat mediate or more viral polyproteins, RNA synthesis in whichassociation are co-translationally and post-translationally processed by virally encodedwith proteases. cellular membranes. Other proteins made by the 44 virus include those that will assemble into viral particles (gray squares). Some of the mature polyprotein processing precursorsThe RdRp mediatesand products the synthesis include of negative- the RNA-dependent RNA polymerase (RdRp; light blue rectangle) andstrand cofactors RNA [( −()RNA]light 45blue antigenome squares (red)) that mediate viral RNA synthesis in association with cellular membranes.using the genome as template. The antigenome is then converted into new (+)RNA genome by the RdRp and then packaged into 46 nascent Other proteins made by the virus include thosevirion that particleswill assemble (gray hexagon). into (b)viral Negative- particles (gray squares). The RdRp mediates the synthesis of negative-strandstrand RNA RNA [( [(−−)RNA])RNA] viruses. antigenome The genomes (red) using the genome as template. of (−)RNA viruses are organized as 47 ribonucleoproteins (RNPs) with their RNA The antigenome is then converted into new (+)RNAmolecules genome (red) bound by by the the RdRp viral polymerase and then packaged into nascent complex (light blue rectangle) and virion particles (gray hexagon). nucleocapsid proteins (N; blue square). 48 Transcription is mediated by the RdRp and cofactor proteins, resulting in the synthesis of 5ʹ capped (purple circle), 3ʹ polyadenylated (AAAAA) mRNAs (green). The mRNAs are Retroelements: Retrons, retroposons, retrotransposons (copia elements in Drosophila, and DIRSI in slime mold) and retroviruses, (HIV, Feline leukaemia retrovirus).

All contain (Rd Dp) in their sequence, which they use to replicate themselves (consequently all possess a high mutation rate: eg HIV, 1x10-4 (1 in 10,000 base pairs replicated incorporate a mutation) vs. 1 x 10 -8 for most human genes. The reproducve cycles of retroviruses and retrotransposons. Retroviruses and Retrotransposons

• Copia elements represent a family of retrotransposons. The number of copia elements depends on the strain of fly and is typically between 20 and 60 copies.

– The element is ~5,000 bp long, with identical direct terminal repeats of 276 bp. – A direct repeat of 5 bp of target DNA is generated at the site of insertion. – There is very little divergence between individual family members; variants usually contain small deletions. – Transcripts of copia are found as abundant poly(A+) mRNAs representing both full-length and part-length transcripts. The reproducve cycles of retroviruses and retrotransposons. Arst's rendering of an electron micrograph showing retroviruses.

© MedicalRF/Photo Researchers, Inc. Retroviruses (HIV) bud from the plasma membrane of an infected cell.

Photos courtesy of Mahew A. Gonda, Ph.D., Partner at Power Ten Medical Ventures I • A retrovirus normally has two copies of its single-stranded RNA genome packaged in the viral particle.

• An integrated provirus is a double- stranded DNA sequence.

• A retrovirus generates a provirus by reverse transcription of the retroviral genome.

RNA --> DNA --> RNA • A retrovirus normally has two copies of its single-stranded RNA genome packaged in the viral particle.

• An integrated provirus is a double- stranded DNA sequence.

• A retrovirus generates a provirus by reverse transcription of the retroviral genome.

RNA --> DNA --> RNA

• A retrovirus normally has two copies of its single-stranded RNA genome packaged in the viral particle.

• An integrated provirus is a double- stranded DNA sequence.

• A retrovirus generates a provirus by reverse transcription of the retroviral genome.

RNA --> DNA --> RNA

RNA

• Retroviral genomes exist as RNA and DNA sequences • A short sequence (R) is repeated at each end of the viral RNA. – The 5′ and 3′ ends are R-U5 and U3-R, respectively. DNA

• Retroviral genomes exist as RNA and DNA sequences • A short sequence (R) is repeated at each end of the viral RNA. – The 5′ and 3′ ends are R-U5 and U3-R, respectively. Integrated DNA

• Retroviral genomes exist as RNA and DNA sequences • A short sequence (R) is repeated at each end of the viral RNA. – The 5′ and 3′ ends are R-U5 and U3-R, respectively. • Reverse transcriptase starts synthesis when a tRNA primer binds to a site 100 to 200 bases from the 5′ end.

• When the enzyme reaches the end, the 5′- terminal bases of RNA are degraded. – This exposes the 3′ end of the DNA product.

• The exposed 3′ end base pairs with the 3′ terminus of another RNA genome.

• Synthesis continues, generating a product in which the 5′ and 3′ regions are repeated. – This gives each end the structure U3-R- U5. • Reverse transcriptase starts synthesis when a tRNA primer binds to a site 100 to 200 bases from the 5′ end.

• When the enzyme reaches the end, the 5′- terminal bases of RNA are degraded. – This exposes the 3′ end of the DNA product.

• The exposed 3′ end base pairs with the 3′ terminus of another RNA genome.

• Synthesis continues, generating a product in which the 5′ and 3′ regions are repeated. – This gives each end the structure U3-R- U5. • Reverse transcriptase starts synthesis when a tRNA primer binds to a site 100 to 200 bases from the 5′ end.

• When the enzyme reaches the end, the 5′- terminal bases of RNA are degraded. – This exposes the 3′ end of the DNA product.

• The exposed 3′ end base pairs with the 3′ terminus of another RNA genome.

• Synthesis continues, generating a product in which the 5′ and 3′ regions are repeated. – This gives each end the structure U3-R- U5. • Reverse transcriptase starts synthesis when a tRNA primer binds to a site 100 to 200 bases from the 5′ end.

• When the enzyme reaches the end, the 5′- terminal bases of RNA are degraded. – This exposes the 3′ end of the DNA product.

• The exposed 3′ end base pairs with the 3′ terminus of another RNA genome.

• Synthesis continues, generating a product in which the 5′ and 3′ regions are repeated. – This gives each end the structure U3-R- U5. • Similar strand switching events occur when reverse transcriptase uses the DNA product to generate a complementary strand. • Strand switching is an example of the copy choice mechanism of recombination. • Similar strand switching events occur when reverse transcriptase uses the DNA product to generate a complementary strand. • Strand switching is an example of the copy choice mechanism of recombination. RNA

DNA

Integrated DNA

• Retroviral genomes exist as RNA and DNA sequences • A short sequence (R) is repeated at each end of the viral RNA. – The 5′ and 3′ ends are R-U5 and U3-R, respectively. Viral DNA Integrates into the Chromosome

• The organization of proviral DNA in a chromosome is the same as a transposon, with the provirus flanked by short direct repeats of a sequence at the target site.

• Linear DNA is inserted directly into the host chromosome by the retroviral enzyme (derived from proteolysis of the pol gene product).

• Two base pairs of DNA are lost from each end of the retroviral sequence during the integration reaction. The organization of proviral DNA in a chromosome is the same as a transposon, with the provirus flanked by short direct repeats of a sequence at the target site.

Linear DNA is inserted directly into the host chromosome by the retroviral integrase enzyme.

•Two base pairs of DNA are lost from each end of the retroviral sequence during the integration reaction.

•Integrase is the only viral protein required for the integration reaction. Replication-defective transforming viruses have a cellular sequence substituted for part of the viral sequence. The defective virus may replicate with the assistance of a helper virus that carries the wild-type functions

75 Replication-defective viruses may be generated through integration and deletion of a viral genome to generate a fused viral–cellular transcript that is packaged with a normal RNA genome. Nonhomologous recombination is necessary to generate the replication-defective transforming genome. 76 Ty elements in yeast generate virus-like parcles. Reprinted from J. Mol. Biol., vol. 292, H. A. AL-Khayat, et al., Yeast Ty retrotransposons..., pp. 65-73. Copyright 1999, with permission from Elsevier [hp:// www.sciencedirect.com/science/journal/00222836]. Photo courtesy of Dr. Hind A. AL- Khayat, Imperial College London, United Kingdom. Ty elements in Yeast (~35 copies per genome) represent a third type of transposositional insertion....Retrotransposition.

Ty elements ~5.1kbp in size and encode for a 300 bp tandem repeats (δ's), which can be seen scattered around Yeast genomes. They cause 5 bp repeats in target site and transpose through an RNA intermediate!!! How can we know this? 78 Ty transposes through a spliced RNA form 79 Ty transposes through a spliced RNA form 80 Ty transposes through a spliced RNA form 81 • There are some retroelements that lack LTRs, and which also transpose via reverse transcriptase -they employ a distinct method of integration and are phylogenetically distinct from both retroviruses and LTR retrotransposons.

• Other elements can be found that were generated by an RNA-mediated transposition event, but they do not themselves code for enzymes that can catalyze transposition.

• Retroelements constitute almost half (48%) of the human genome. • Retrotransposons of the viral superfamily are transposons that mobilize via an RNA that does not form an infectious particle. • Some retrotransposons directly resemble retroviruses in their use of LTRs. Others do not, and have no LTRs.

• Despite having an RT activity, LINES lack the LTRs of the viral superfamily and use a unique mechanism to prime the reverse transcription rxn. • The non-viral superfamily may have originated from RNA sequences; • SINES are derived from RNA polymerase III transcripts. • LINES and SINES comprise a major part of the animal genome. They were originally defined by the existence of a large number of relatively short sequences that were related to one another.

• They are described as interspersed elements because of their common occurrence and widespread distribution. L1 = active human LINES; ALU = active human SINES • short-interspersed elements (SINEs) – A major class of short (<500 bp) nonautonomous retrotransposons that occupy ~13 -15% of the human genome. • Alu element – One of a set of dispersed, related sequences, each ~300 bp long, in the human genome (members of the SINE family). Karyotype from a female human lymphocyte (46, XX). Chromosomes were hybridized with a probe for Alu elements (green) and counterstained with TOPRO-3 (red). Alu elements were used as a marker for chromosomes and chromosome bands rich in genes. • DNA transposons and LTR elements are believed to be “extinct” in the human genome, but the average human carries approximately 80 – 100 potentially active L1 elements in a diploid genome. • LINES and SINES, however, are NOT extinct • The full length human L1 retrotransposon is 6kb and contains – a 910 bp 5’-UTR with bidirectional promoter activity – An ORF1 region which encodes an RN binding protein with a leucine zipper domain – An ORF2 region which encodes a 150 kDa protein with endonuclease and reverse transcriptase activities – A 3’-UTR which contains a functional sequence • The L1 element is flanked by 2 to 20 bp target site duplications.

• The EN domain is thought to originate from a host endonuclease present in early eukaryotes. The element can move without a functional EN, but the endonuclease-independent integration is less efficient and occurs rarely. L1 expression leads to different types of DNA damage.

Schematic structures of an SVA element (labeled SVA), showing the CCCTCT repeat, the Alu derived (A-like) region, the variable number tandem repeat (VNTR) region, and the long terminal repeat (LTR)derived region; an Alu element (labeled Alu (SINE)), showing left (purple) and right (pink) halves separated by the Arich region (A) and the variable length Atail ((A)n) followed by the 3’ region (white), which has a variable length and sequence; and an L1 element (labeled LINE1), showing open reading frame (ORF)1 (light blue) and ORF2 (dark blue) and the 5’ untranslated region, interORF region and 3’ untranslated region (white).

(a) The typical insertion of these elements into the genome, which can lead to insertional mutagenesis. In breast cancer BRCA1 and BRCA2 are also known to be disrupted by TE insertion

(b) Dispersed repetitive elements such as Alu elements can undergo non-allelic homologous recombination, which can cause a deletion (shown) or duplication (not shown). The dashed arrow indicates the potential site of DNA damage by an L1 endonuclease that may help initiate these recombination events.

(c) Potential outcomes of the repair of the L1 induced double strand breaks (DSBs). The L1 recognition site is in black; surrounding sequence is in blue; inserted nucleotides are in red. The associated changes are typical of what might be seen with repair of the DSB by non homologous end joining (NHEJ) mechanisms. It is also possible that the sites are simply re-ligated with no mutation occurring, or alternatively, these sites may cause recombination, as shown in (b). Belancio et al.: Genome Medicine 2009, 1:97 2005: Callinan Pauline A; Wang Jianxin; Herke Scott W; Garber Randall K; Liang Ping; Batzer Mark A Alu retrotransposition-mediated deletion. Journal of molecular biology 2005;348(4):791-800 A two-exon gene is flanked by two Alu elements and a neighbouring replication termination site. Recombination between the two Alu elements leads to a tandem duplication event, as does a replication error instigated by the replication termination site.

Retrotransposition of the mRNA of the gene leads to the random integration of an intron-less paralogue at a distinct genomic location.