Editing Chloroplast (section)

===DNA repair and replication===

In chloroplasts of the moss ''[[Physcomitrella patens]]'', the [[DNA mismatch repair]] protein Msh1 interacts with the [[homologous recombination|recombination]]al [[DNA repair|repair]] proteins [[RecA]] and RecG to maintain chloroplast [[genome]] stability.<ref name="Odahara-2017">{{cite journal | vauthors=Odahara M, Kishita Y, Sekine Y | title=MSH1 maintains organelle genome stability and genetically interacts with RECA and RECG in the moss Physcomitrella patens | journal=The Plant Journal | volume=91 | issue=3 | pages=455–465 | date=August 2017 | pmid=28407383 | doi=10.1111/tpj.13573 | doi-access=free }}</ref> In chloroplasts of the plant ''[[Arabidopsis thaliana]]'' the [[RecA]] protein maintains the integrity of the chloroplast's DNA by a process that likely involves the recombinational repair of [[DNA damage (naturally occurring)|DNA damage]].<ref name="Rowan-2010">{{cite journal | vauthors=Rowan BA, Oldenburg DJ, Bendich AJ | title=RecA maintains the integrity of chloroplast DNA molecules in Arabidopsis | journal=Journal of Experimental Botany | volume=61 | issue=10 | pages=2575–88 | date=June 2010 | pmid=20406785 | pmc=2882256 | doi=10.1093/jxb/erq088 }}</ref>

[[File:CpDNA Replication.png|thumb|upright=1.65|Chloroplast DNA replication via multiple [[D-loop]] mechanisms. Adapted from Krishnan NM, Rao BJ's paper "A comparative approach to elucidate chloroplast genome replication."]]
The mechanism for chloroplast DNA (cpDNA) replication has not been conclusively determined, but two main models have been proposed. Scientists have attempted to observe chloroplast replication via [[electron microscopy]] since the 1970s.<ref name="Krishnan-2009">{{cite journal | vauthors=Krishnan NM, Rao BJ | title=A comparative approach to elucidate chloroplast genome replication | journal=BMC Genomics | volume=10 | issue=237 | page=237 | date=May 2009 | pmid=19457260 | pmc=2695485 | doi=10.1186/1471-2164-10-237 | doi-access=free }}</ref><ref name="Heinhorst-1993">{{cite journal | vauthors=Heinhorst S, Cannon GC |title=DNA replication in chloroplasts|journal=Journal of Cell Science|date=1993|volume=104|pages=1–9|doi=10.1242/jcs.104.1.1|url=https://aquila.usm.edu/cgi/viewcontent.cgi?article=7560&context=fac_pubs}}</ref> The results of the microscopy experiments led to the idea that chloroplast DNA replicates using a double displacement loop (D-loop). As the D-loop moves through the circular DNA, it adopts a theta intermediary form, also known as a Cairns replication intermediate, and completes replication with a rolling circle mechanism.<ref name="Krishnan-2009"/><ref name="Bendich-2004">{{cite journal |vauthors=Bendich AJ |date=July 2004 |title=Circular chloroplast chromosomes: the grand illusion |journal=The Plant Cell |volume=16 |issue=7 |pages=1661–6 |doi=10.1105/tpc.160771 |pmc=514151 |pmid=15235123|bibcode=2004PlanC..16.1661B }}</ref> Transcription starts at specific points of origin. Multiple replication forks open up, allowing replication machinery to transcribe the DNA. As replication continues, the forks grow and eventually converge. The new cpDNA structures separate, creating daughter cpDNA chromosomes.

In addition to the early microscopy experiments, this model is also supported by the amounts of [[deamination]] seen in cpDNA.<ref name="Krishnan-2009"/> Deamination occurs when an amino group is lost and is a mutation that often results in base changes. When adenine is deaminated, it becomes [[hypoxanthine]]. Hypoxanthine can bind to cytosine, and when the XC base pair is replicated, it becomes a GC (thus, an A → G base change).<ref name=Biocyclopedia>{{cite web|title=Effect of chemical mutagens on nucleotide sequence|url=http://www.biocyclopedia.com/index/genetics/mutations_molecular_level_mechanism/effect_of_chemical_mutagens_on_nucleotide_sequence.php|website=Biocyclopedia|access-date=24 October 2015}}</ref>
[[File:Adenine Deaminates to Guanine.png|thumb|left|upright=1.35|Over time, base changes in the DNA sequence can arise from deamination mutations. When adenine is deaminated, it becomes hypoxanthine, which can pair with cytosine. During replication, the cytosine will pair with guanine, causing an A --> G base change.]]

In cpDNA, there are several A → G deamination gradients. DNA becomes susceptible to deamination events when it is single stranded. When replication forks form, the strand not being copied is single stranded, and thus at risk for A → G deamination. Therefore, gradients in deamination indicate that replication forks were most likely present and the direction that they initially opened (the highest gradient is most likely nearest the start site because it was single stranded for the longest amount of time).<ref name="Krishnan-2009"/> This mechanism is still the leading theory today; however, a second theory suggests that most cpDNA is actually linear and replicates through homologous recombination. It further contends that only a minority of the genetic material is kept in circular chromosomes while the rest is in branched, linear, or other complex structures.<ref name="Krishnan-2009"/><ref name="Bendich-2004"/>

One of competing model for cpDNA replication asserts that most cpDNA is linear and participates in [[homologous recombination]] and replication structures similar to the linear and circular DNA structures of [[bacteriophage T4]].<ref name="Bendich-2004"/><ref name="Bernstein-1973">{{cite journal | vauthors=Bernstein H, Bernstein C | title=Circular and branched circular concatenates as possible intermediates in bacteriophage T4 DNA replication | journal=Journal of Molecular Biology | volume=77 | issue=3 | pages=355–61 | date=July 1973 | pmid=4580243 | doi=10.1016/0022-2836(73)90443-9 }}</ref> It has been established that some plants have linear cpDNA, such as maize, and that more species still contain complex structures that scientists do not yet understand.<ref name="Bendich-2004"/> When the original experiments on cpDNA were performed, scientists did notice linear structures; however, they attributed these linear forms to broken circles.<ref name="Bendich-2004"/> If the branched and complex structures seen in cpDNA experiments are real and not artifacts of concatenated circular DNA or broken circles, then a D-loop mechanism of replication is insufficient to explain how those structures would replicate.<ref name="Bendich-2004"/> At the same time, homologous recombination does not expand the multiple A --> G gradients seen in plastomes.<ref name="Krishnan-2009"/> Because of the failure to explain the deamination gradient as well as the numerous plant species that have been shown to have circular cpDNA, the predominant theory continues to hold that most cpDNA is circular and most likely replicates via a D loop mechanism.