Direct pairing of homologous DNA double helices may involve the B-to-C form transition

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TITLE
Direct pairing of homologous DNA double helices may involve the B-to-C form transition
AUTHORS
Alexey K. Mazur1,2 and Eugene Gladyshev2
1. CNRS, Université Paris Cité, UPR 9080, Laboratoire de Biochimie Théorique, 13 rue Pierre et Marie
Curie, Paris, France
2. Institut Pasteur, Université Paris Cité, Group Fungal Epigenomics, Paris, France
Correspondence:
alexey@ibpc.fr (A.K.M)
eugene.gladyshev@gmail.com (E.G)
KEYWORDS
Recombination-independent, homology recognition, C-DNA
ABSTRACT
In many organisms, homologous (or repetitive) chromosomal regions can associate or/and undergo concerted
epigenetic changes in the absence of DNA breakage and recombination. The direct specific pairing of DNA
duplexes with similar nucleotide sequences represents an attractive mechanism for recognizing such regions.
Whereas the pairing of B-DNA duplexes may involve a large energy barrier, C-DNA duplexes are expected
to pair much more readily. This unique feature of C-DNA is largely due to the fact that its major groove is
wide and very shallow, permitting almost perfect initial homologous contacts between two duplexes without
clashing. Overall, the conjectured role of C-DNA in recombination-independent pairing should revive the
efforts to understand its structure and function in the cell.
HIGHLIGHTS
Homologous (or repetitive) chromosomal regions can engage in pairing or/and undergo concerted
epigenetic changes in the apparent absence of DNA breakage and recombination.
The direct recognition and pairing of homologous double-stranded DNA (dsDNA) molecules repre-
sents an attractive underlying mechanism.
Direct dsDNA-dsDNA pairing during premeiotic and meiotic silencing in filamentous fungi arguably
involves segments of C-DNA.
The C-DNA conformation is particularly suitable for the direct homologous pairing.
The B-to-C DNA transition may serve as a low-energy pathway during recombination-independent
DNA homology search and recognition.
OUTSTANDING QUESTIONS
How can C-DNA be reliably detected and quantified in vitro and in vivo?
Which factors could promote the B-to-C DNA transition in vivo?
Can C-DNA exist prior to the pairing?
Alternatively, does the B-to-C DNA transition only occur concomitantly with the pairing?
1
Recombination-independent homologous pairing: its existence and implications
In many organisms, homologous (or repetitive) chromosomal regions can engage in pairing or/and undergo
concerted epigenetic changes in the apparent absence of DNA breakage and recombination [1]. A large
number of such homology-dependent phenomena has been described in animals [1,2]. For example, during
mammalian development, selected allelic loci are known to pair transiently, presumably to establish appro-
priate patterns of gene expression [3]. The association of X chromosomes prior to random X-chromosome
inactivation in mammalian females provides a paradigmatic example of such a process [4]. Moreover, mam-
malian genomes contain large amounts of repetitive DNA normally silenced in the form of constitutive hete-
rochromatin [5]. The initiation of such silencing can occur on newly introduced exogenous tandem repeat
arrays and does not require RNA interference [6,7], whereas its pathological misregulation has been impli-
cated in several types of cancer [8] and other disease, such as Type I Facioscapulohumeral muscular dys-
trophy [9]. The mechanistic basis of homology (or repeat) recognition in all these situations remains
unknown.
The classical example of recombination-independent pairing was described in Drosophila melanogaster and
other Diptera insects [10]. In these animals, homologous chromosomes remain associated in the majority of
cell types during development and later in the adult life. The paired state is dynamic [11] and plays a critical
role in regulating transvection, a phenomenon in which two alleles comprise one expression unit due to their
close physical proximity [10]. Recent studies using haplotype-resolved HiC [12,13] and super-resolution
microscopy [14] yielded two important insights. First, it became apparent that the degree of pairing fluctu-
ates substantially along the chromosome lengths, where tightly-paired regions are interrupted by loosely-
paired regions. Second, within the tightly-paired regions, homologous segments could not be distinguished
even at the highest resolution, hinting at a possibility that such pairing was established at distances corre-
sponding to direct DNA-DNA contacts.
The widespread occurrence of recombination-independent homology-directed phenomena contrasts with the
limited understanding of their basis for at least two reasons. First, such processes normally involve large and
functionally important genomic regions, which are hard to manipulate and analyze experimentally. Second,
the only currently accepted general mechanism of homology recognition between double-stranded DNA
(dsDNA) is the Watson-Crick base pairing in cross-hybridization of complementary single strands, which
occurs normally after strand breaking and base-pair opening by enzymes involved in recombination. Thus,
elucidating mechanistic aspects of recombination-independent pairing requires both better model systems
and better understanding of the biophysical properties of DNA.
Meiosis as a dedicated pairing stage
Homologous pairing is most apparent in meiosis, a specialized cell division that halves the number of chro -
mosomes to generate gametes [15]. Observable meiotic pairing commences in early prophase I, normally
after chromosomes begin to compact and individualize [16]. By late prophase I, homologous chromosomes
become closely aligned in a configuration known as synapsis.
Canonically, the synapsis of homologous chromosomes requires the break-generating function of Spo11 [17].
This program is documented in mammals and also in some popular model organisms such as S. cerevisiae
[18]. Alternatively, homologous chromosomes can pair and fully synapse in the absence of Spo11 [18]. Such
pairing takes place in D. melanogaster and the roundworm Caenorhabditis elegans [18]. It is called recombi-
nation-independent because it occurs normally in the absence of Spo11-mediated double-strand breaks [16].
Several hypotheses were put forward to explain the phenomenon of recombination-independent pairing. For
example, the pairing was proposed to occur directly at the DNA level via G-quadruplexes [19], other four-
2
stranded DNA structures [20,21], or long-range electrostatic interactions [22]. Alternatively, the pairing was
suggested to take place at the level of chromosomal domains relying on the indirect contacts mediated by
proteins [23], non-coding RNAs [24] as well as large-scale genomic features such as centromeres, telomeres
[25,26] and regions of active transcription [27].
The questions regarding the nature of recombination-independent pairing in meiosis proved non-trivial in
part because the behavior of meiotic chromosomes is normally studied by tracing the chromosomal axes and
the synaptonemal complex [28], which probably start to develop only after the early recombination-indepen-
dent pairing has peaked.
MSUD as a sensitive readout of the early recombination-independent homologous pairing in meiosis
A meiotic process has been described in a number of filamentous fungi in which unpairable allelic sequences
trigger RNAi-mediated silencing in prophase I [29–31]. This process was named “meiotic silencing by
unpaired DNA”, MSUD [30]. Pioneering studies in Neurospora crassa defined some basic properties of
unpaired DNA capable of inducing MSUD, including the threshold length of ~1 kbp and the absence of a
requirement for promoter activity [30]. Collective efforts of several research groups uncovered many genes
involved in MSUD [30], yet the mechanism by which unpairable DNA can be detected remained unknown.
Two aspects of the meiotic program in N. crassa and related fungi attest to the high efficiency of MSUD and
the associated homology-recognition mechanism. First, these organisms have a haploid premeiotic stage, in
which parental nuclei undergo the last round of DNA replication and fuse right before the onset of meiosis
[28,32]. Second, the early signs of MSUD (i.e., phenotypic loss-of-function of an unpaired gene) can be
detected shortly after the fusion of the haploid nuclei [31]. Yet MSUD still operates normally in the absence
of Spo11 and Rad51/Dmc1 proteins, when all meiotic recombination has been essentially eliminated [33].
The fact that MSUD is independent from Spo11 and Rad51/Dmc1 contrasts with the fact that in N. crassa the
synapsis of homologous chromosomes actually requires Spo11 [34,35]. Thus, while N. crassa shares this key
features of its meiotic program with other canonical” systems, it also features a transient recombination-
independent process, supporting the possibility that the latter can be a part of the canonical meiotic program
as well. Indeed, transient Spo11-independent pairing has been reported in mice [23,36] and yeast [37,38].
Whether this transient pairing is requited for the Spo11-dependent pairing and synapsis remains an open
question.
MSUD shares its basis of homology recognition with repeat-induced point mutation (RIP)
In some filamentous fungi premeiotic nuclei can engage in two closely-related processes known as “repeat-
induced point mutation” (RIP) and “methylation induced premeiotically” (MIP) [39,40]. During RIP and
MIP, gene-sized repeats of genomic DNA become subject to strong cytosine-to-thymine mutation (RIP) or
cytosine methylation (MIP). The capacity of RIP to recognize repeats irrespective of their relative and abso -
lute positions in the genome (i.e., being on the same or two different chromosomes) as well as their origin,
sequence composition and coding potential suggests that a general and very efficient homology search is
involved [40].
Studies in N. crassa have found that RIP, like MSUD, recognizes repeats by a recombination-independent
mechanism [41]. They further uncovered the ability of RIP to detect interspersed homology, i.e., pairs of
sequences that share periodically occurring short regions of matching base-pairs interleaved with longer
stretches of mismatching base-pairs, over the total length of several hundred base-pairs [41]. Remarkably,
interspersed homologies containing matching regions of only 4-bp and corresponding to the overall sequence
identify of only 36% can still be recognized by RIP [42].
3
摘要:

TITLEDirectpairingofhomologousDNAdoublehelicesmayinvolvetheB-to-CformtransitionAUTHORSAlexeyK.Mazur1,2andEugeneGladyshev21.CNRS,UniversitéParisCité,UPR9080,LaboratoiredeBiochimieThéorique,13ruePierreetMarieCurie,Paris,France2.InstitutPasteur,UniversitéParisCité,GroupFungalEpigenomics,Paris,FranceCor...

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